Carbohydrate oxidase and use thereof in baking

ABSTRACT

The properties of dough or bread can be improved by the addition of a carbohydrate oxidase which can oxidize the reducing end of an oligosaccharide more efficiently than the corresponding monosaccharide, e.g., preferentially oxidizing maltodextrins or cellodextrins over glucose. A novel carbohydrate oxidase having the capability to oxidize maltodextrins and cellodextrins more efficiently than glucose may be obtained from a strain of Microdochium, particularly M. nivale. The amino acid sequence of the novel carbohydrate oxidase has very low homology (&lt;20% identity) with known amino acid sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119 of Danishapplications PA 1997 01505 filed Dec. 22, 1997 and PA 1998 00763 filedJun. 4, 1998, and of U.S. provisional application No. 60/068,717 filedDec. 23, 1997 and provisional application No. 60/088,725 filed Jun. 10,1998, the contents of which are fully incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to the use in baking of a carbohydrateoxidase and to a novel carbohydrate oxidase.

DESCRIPTION OF THE RELATED ART

In the bread-making process it is known to add bread-improving and/ordough-improving additives to the bread dough, the action of which, interalia, results in improved texture, volume, flavor and freshness of thebread as well as improved machinability and stability of the dough.

Dough "conditioners" to strengthen the gluten and improve therheological and handling properties of the dough are well known in theindustry and have long been used. Nonspecific oxidants, such as iodates,peroxides, ascorbic acid, potassium bromate and azodicarbonamide have agluten strengthening effect. It has been suggested that theseconditioners induce the formation of interprotein bonds which strengthenthe gluten, and thereby the dough.

It is also known to use glucose oxidase to strengthen the gluten andimprove the rheological and handling properties of the dough. Thus, U.S.Pat. No. 2,783,150 discloses the use of glucose oxidase in flour toimprove dough strength, and texture and appearance of baked bread. EP321 811 and EP 338 452 disclose the use in baking of glucose oxidase incombination with other enzymes (sulfhydryl oxidase, hemicellulase,cellulase). However, the effectiveness of glucose oxidase as a doughand/or bread improving additive is limited due to the generally lowglucose content in cereal flours used in the preparation of bakedproducts.

Thus there has been interest in identifying oxidoreductases which act onsubstrates other than glucose. WO 96/39851 discloses the use of a hexoseoxidase which is capable of oxidizing D-glucose and several otherreducing sugars including maltose, lactose, galactose, xylose, arabinoseand cellobiose to their respective lactones with a subsequent hydrolysisto the respective aldobionic acids. WO 97/22257 discloses the use of apyranose oxidase in baking. The enzyme catalyses the oxidation ofseveral monosaccharides at position C2 with the concomitant release ofhydrogen peroxide. Although glucose in its pyranose form tends to be thepreferred substrate, the enzyme is capable of oxidizing othersubstrates, e.g., furanoses, such as xylose.

Although enzymes that catalyze the oxidation of glucose and other sugarsdirectly to the corresponding aldonic acids appear to be widelydistributed in nature, most of the known sugar oxidases are specific tomonosaccharides. An oligosaccharide oxidase, isolated and purified fromwheat bran culture of a soil-isolated Acremonium strictum strain T1, hasbeen described by Lin, et al, (1991, Biochim. Biophys. Acta 1118:41-47).The enzyme has the capability of oxidizing oligosaccharides with aglucose residue on the reducing end. The enzyme demonstrated reactivitytoward maltose, lactose, cellobiose and maltooligosaccharides composedof up to seven glucose units. JP-A 5-84074 discloses use of the enzymeas an analytical reagent.

SUMMARY OF THE INVENTION

The inventors have found that the properties of dough or bread can beimproved by the addition of a carbohydrate oxidase which can oxidize thereducing end of an oligosaccharide more efficiently than thecorresponding monosaccharide, e.g., preferentially oxidizingmaltodextrins or cellodextrins over glucose. This can lead to improvedfirmness, stickiness, stability and robustness of the dough. It can alsoincrease the tolerance of the dough towards increased mixing time,fermentation time and water content.

The inventors have also found a novel carbohydrate oxidase with thecapability to oxidize maltodextrins and cellodextrins more efficientlythan glucose. The novel oxidase may be obtained form Microdochium,particularly M. nivale. The inventors have isolated and deposited such astrain as M. nivale CBS 100236. The amino acid sequence of the novelcarbohydrate oxidase has very low homology (<20% identity) with knownamino acid sequences.

Accordingly, the invention provides a process for preparing a doughand/or a baked product made from a dough comprising adding to the dougha carbohydrate oxidase which has a higher activity on an oligosaccharidehaving a degree of polymerization of 2 or higher as a substrate than onthe corresponding monosaccharide. The invention also provides abread-improving additive comprising the carbohydrate oxidase. Thebread-improving additive may comprise a second enzyme (amylase,cellulase, hemicellulase, lipase or phospholipase), and it may be inagglomerated powder or granulated form.

The invention further provides a novel carbohydrate oxidase. Thecarbohydrate oxidase may be a polypeptide produced by Microdochiumnivale CBS 100236 or having an amino acid sequence as shown in SEQ IDNO: 2, or it may be an analogue thereof. The carbohydrate oxidase mayalso be derivable from a strain of Microdochium and have an oxidizingactivity on maltotetraose which is at least twice as much as theoxidizing activity on glucose at a substrate concentration of 0.83 mM.

The invention also provides a method of producing said carbohydrateoxidase by cultivation of Microdochium. The invention further provides anucleic aid construct comprising a nucleic acid sequence encoding thecarbohydrate oxidase of the invention, recombinant expression vectorsand recombinant host cells which are advantageously used in therecombinant production of the carbohydrate oxidase of the presentinvention. In yet a further aspect, the present invention providesrecombinant methods for producing a carbohydrate oxidase of theinvention comprising cultivating a host cell under conditions conduciveto the production of the carbohydrate oxidase and recovering thecarbohydrate oxidase from the cells and/or culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 illustrate plasmids pBANe15, pEJG33 and pEJG35, respectively.Details are given in the Examples.

DETAILED DESCRIPTION OF THE INVENTION

Use of Carbohydrate Oxidase in Baking

The present invention provides the addition to dough of a carbohydrateoxidase which has a higher activity on an oligosaccharide having adegree of polymerization of 2 or higher as a substrate than on thecorresponding monosaccharide. The carbohydrate oxidase may be added inthe form of a dough and/or bread-improving additive as described below.

The carbohydrate oxidase is generally added in amount which is effectivefor providing a measurable effect on at least one property of interestof the dough and/or baked product. The bread-improving and/or doughimproving additive is generally included in the dough in an amountcorresponding to 0.01-5%, in particular 0.1-3%. The enzyme is typicallyadded in an amount corresponding to 0.01-100 mg enzyme protein per kg offlour, preferably 0.1-25 mg per kg, more preferably 0.1-10 mg per kg,and most preferably 0.5-5 mg per kg.

The level of oligosaccharides in dough can be increased by the additionof an amylase which hydrolyzes starch to form oligosaccharides as a mainproduct, e.g., a Bacillus stearothermophilus maltogenic alpha-amylase(commercially available as Novamyl®), an Aspergillus oryzaealpha-amylase (commercially available as Fungamyl®) or a beta-amylase.

The use of an oligosaccharide oxidase may result in an increased volumeand an improved crumb structure and softness of the baked product, aswell as an increased strength, stability and reduced stickiness of thedough, thus resulting in improved machinability. The effect may be inaddition to, or as a consequence of a gluten strengthening effect whichis discussed below. The effect on the dough may be particularlyadvantageous when a poor quality flour is used. The improvedmachinability is of particular importance in connection with dough whichis to be processed industrially.

Dough stability is one of the most important characteristics of a bakingdough and is important for both large scale and small scaleapplications. A stable, or strong, dough is capable of a greatertolerance of mixing time, proofing time and of mechanical vibrationsduring dough transport, whereas a weak, or less stable, dough is lesstolerant to these treatments. Whereas flour with a high gluten contentand a good gluten quality contribute to a strong dough, flour containinga low protein content or with poor gluten quality results in a weakdough. Thus, a strong dough which has superior rheological and handlingproperties results from flour containing a strong gluten network.

The oligosaccharide oxidase may be added to any mixture of doughsubstances, to the dough, or to any of the substances to be included inthe dough; that is, the oligosaccharide oxidase may be added in any stepof the dough preparation and may be added in one, two or more steps,where appropriate and avoiding exposure of the enzyme to strongchemicals or conditions where it could become inactivated.

Substrate Specificity

The carbohydrate oxidase preferably has a higher activity on amaltooligosaccharide having a degree of polymerization of 2-6(particularly maltose, maltotriose or maltotetraose) than on glucose ata substrate concentration of 10 mM or less. The comparison may be madeat a substrate concentration of 1 mM or less, and the activity onmaltotetraose is preferably more than twice of the activity on glucose.The carbohydrate oxidase may have an oxidizing activity on maltodextrinsor cellodextrins maltotetraose which is at least two times the oxidizingactivity on glucose at a substrate concentration of 0.83 mM.

Such substrate concentrations are representative of the concentration intypical doughs prepared according to usual baking practice. Thus, forexample, in an extract made from a dough the concentration of maltosewas found to be 4.1 mM, which corresponds to 41 mmoles/kg dough obtainedfrom a 1:10 extraction for 1 hour at 40° C. as described by Poulsen, C.,et al (1996. Cereal Chem., 75: 51-57). It was further mentioned that theamount of extractable maltose could be higher if sufficient endogenousamylolytic activity (e.g., beta-amylase) was present in the flour, orexogenous amylolytic enzymes was added to the dough or flour, as isoften the practice. WO 96/39851 similarly discloses that maltose ispresent in dough at a level of 1.4% (w/w). Thus, the amount of availablesubstrate, e.g., maltose, can differ depending on flour type andquality, recipe, mixing and fermentation process, as well as on thepresence of other additives.

At pH 6 and 50 mM, a preferred carbohydrate oxidase from M. nivale hasthe following preference (descending order):cellobiose>maltose>glucose>xylose>lactose. Based on Michaeli-Mentenkinetics, the apparent K_(m) values for the preferred substrates are: 59mM (cellobiose), 11 mM (maltose), 42 mM (glucose); V_(max) is similarfor glucose and maltose. Thus, the oxidase shows preference for maltoseover glucose, particularly at low substrate concentrations (below 10mM).

A preferred carbohydrate oxidase from M. nivale is capable of oxidizingoligosaccharides having a degree of polymerization (DP) of DP2-DP5, at asubstrate concentration of 0.83 mM at a higher rate than thecorresponding monosaccharide. Thus, the enzyme can hydrolyze bothmaltodextrins and cellodextrins wherein the monosaccharide units arelinked by alpha-1,4 or beta-1,4 glucosidic bonds, respectively, at ahigher rate than glucose. The carbohydrate oxidase can hydrolyze allcellodextrins having DP2-DP5 equally well and at a level around 10-foldhigher than the monosaccharide glucose. With maltodextrins as thesubstrate, the activity of the carbohydrate oxidase ranged from11/2-fold higher for maltohexaose to almost 5-fold higher formaltotetraose than for the monosaccharide.

Carbohydrate Oxidase Properties

The carbohydrate oxidase is preferably active and stable at a pH in therange of 5-7, e.g. having more than 40% activity in this range, and mostpreferably having optimum activity in this range. A preferredcarbohydrate oxidase from M. nivale has optimum activity around pH 6 andshows an activity which is at least 80% (relative to the maximumactivity) in the pH range 5-7. At 40° C., it is stable in the pH range4-9, but unstable at pH 3.

The carbohydrate oxidase is preferably active and stable at 20-45° C.,e.g. having more than 50% activity in this range, and most preferablyhaving optimum activity in this range. A preferred carbohydrate oxidasefrom M. nivale has optimum activity around 40° C. and exhibits at least70% activity (relative to maximum activity) in the range 30-60° C. At pH6, it is stable up to 60° C., but inactivated at 70° C. It has adenaturation temperature of 73° C.

The carbohydrate oxidase is able to oxidize reducing oligosaccharideswith a glucose residue on the reducing end. It oxidizes the glucoseresidue at the 1-position to form the corresponding acid. Thus, itoxidizes maltose to form maltobionic acid and lactose to formlactobionic acid.

The carbohydrate oxidase activity may be isolated, e.g. essentially freeof other non-carbohydrate oxidase polypeptides, for example, more thanabout 80% pure, and more preferably more than about 90% pure on aprotein basis as determined by SDS-PAGE.

A preferred carbohydrate oxidase from M. nivale has a molecular weightof approximately 52 kDa as determined by SDS-PAGE and an isoelectricpoint of approximately 8.9. It shows dehydrogenase as a side activitywith electron acceptors such as potassium ferricyanide, methylene blue,benzoquinone and 2,6-dichlorophenol-indophenol (DCPIP).

Sources of Carbohydrate Oxidase

The oligosaccharide oxidase may be obtained from a microbial source,such as a fungus, e.g., a filamentous fungus or a yeast, in particularan Ascomycota fungus, e.g. Euascomycetes, especially Pyrenomycetes.

The carbohydrate oxidase may be derived, e.g., from a mitosporicPyrenomycetes such as Acremonium, in particular, A. strictum, such asATCC 34717 or T1; A. fusidioides, such as IFO 6813; or A. potronii, suchas IFO 31197. In a preferred embodiment, the oligosaccharide oxidase isobtained from the source disclosed by Lin, et al, (1991, Biochim.Biophys. Acta 1118:41-47) and in JP-A 5-84074.

The carbohydrate oxidase may further be obtained from microorganisms ofXylariales; especially mitosporic Xylariales such as the genusMicrodochium, particularly the species M. nivale. Such strains arereadily accessible to the public in culture collections, such as theAmerican Type Culture Collection (ATCC), Deutsche Sammlung vonMikroorganismen und Zelikulturen GmbH (DSM) and Centraalbureau VoorSchimmelcultures (CBS).

The genus Microdochium is described in Microdochium Syd (Samuels andHallett, 1983, TBMS 81:473). Some strains of Microdochium have beendescribed under the synonyms Gerlachia, G. nivalis, G. oryzae, Fusariumnivale or Rynchosporium oryzae. They are further described byMonographella (Hyponectr) fide (Muller, 1977, Rev. mycol. 41:129).

A preferred strain is M. nivale, NN008551. This was isolated fromnatural sources taken in India and deposited according to the BudapestTreaty on the International Recognition of the Deposits ofMicroorganisms for the Purpose of Patent Procedures on Dec. 4, 1997 atthe Centraalbureau voor Schimmelcultures under Accession No. CBS 100236.

The inventors have isolated the gene encoding the carbohydrate oxidasefrom M. nivale CBS 100236 and inserted it into E. coli. The E. colistrain harboring the gene was deposited according to the Budapest Treatyon the International Recognition of the Deposits of Microorganisms forthe Purpose of Patent Procedures on Jun. 12, 1998 at the AgriculturalResearch Service Collection (NRRL), 1815 North University Street,Peoria, Ill., and designated NRRL B-30034.

Additional Enzyme

The carbohydrate oxidase may be added to the dough as the only enzyme,or it may be used in combination with one or more additional enzymes.The additional enzyme may be an amylase (e.g. as described above), acyclodextrin glucanotransferase, a peptidase, in particular, anexopeptidase, a transglutaminase, a lipase, a phospholipase, acellulase, a hemicellulase, in particular a pentosanase such asxylanase, a protease, a protein disulfide isomerase, e.g., a proteindisulfide isomerase as disclosed in WO 95/00636, a glycosyltransferase,and an oxidoreduc-tase, e.g., a peroxidase, a laccase, a glucoseoxidase, a pyranose oxidase, a lipoxygenase, an L-amino acid oxidase oran additional carbohydrate oxidase, and the like.

The additional enzyme may be of any origin, including mammalian andplant, and preferably of microbial (bacterial, yeast or fungal) originand may be obtained by techniques conventionally used in the art.

The amylase may be derived from a bacterium or a fungus, in particularfrom a strain of Aspergillus, preferably a strain of A. niger or A.oryzae, or from a strain of Bacillus. Some examples are alpha-amylase,e.g. from Bacillus amyloliquefaciens, and amyloglucosidase, e.g. from A.niger. Commercial products include BAN and AMG (products of Novo NordiskA/S, Denmark), Grindamyl A 1000 or A 5000 (available from GrindstedProducts, Denmark) and Amylase H and Amylase P (products ofGist-Brocades, The Netherlands).

The protease may be Neutrase (available from Novo Nordisk A/S, Denmark).

The lipase may be derived from a strain of Thermomyces (Humicola),Rhizomucor, Candida, Aspergillus, Rhizopus, or Pseudomonas, inparticular from T. lanuginosus (H. lanuginosa, EP 305,216), Rhizomucormiehei (EP 238,023), C. antarctica (e.g. Lipase A or Lipase B describedin WO 88/02775), A. niger, Rhizopus delemar or Rhizopus arrhizus or P.cepacia (EP 214,761 and WO 89/01032).

Dough

The dough is generally a flour dough comprising wheat meal or wheatflour and/or other types of meal, flour or starch such as corn flour,corn starch, rye meal, rye flour, oat flour, oat meal, soy flour,sorghum meal, sorghum flour, rice starch, rice flour, potato meal,potato flour or potato starch.

The dough may be fresh, frozen or par-baked.

The dough is normally a leavened dough or a dough to be subjected toleavening. The dough may be leavened in various ways, such as by addingchemical leavening agents, e.g., sodium bicarbonate or by adding aleaven (fermenting dough), but it is preferred to leaven the dough byadding a suitable yeast culture, such as a culture of Saccharomycescerevisiae (baker's yeast), e.g. a commercially available strain of S.cerevisiae.

The dough may also comprise other conventional dough ingredients, e.g.:proteins, such as milk or milk powder, gluten, and soy; eggs (eitherwhole eggs, egg yolks or egg whites); shortening such as granulated fator oil; an oxidant such as ascorbic acid, potassium bromate, potassiumiodate, azodicarbonamide (ADA) or ammonium persulfate; a reducing agentsuch as L-cysteine; a sugar; a salt such as sodium chloride, calciumacetate, sodium sulfate or calcium sulfate. The dough may furthercomprise an emulsifier such as mono- or diglycerides, diacetyl tartaricacid esters of mono- or diglycerides, sugar esters of fatty acids,polyglycerol esters of fatty acids, lactic acid esters ofmonoglycerides, acetic acid esters of monoglycerides, polyoxyethylenestearates, phospholipids, lecithin and lysolecithin.

The dough may be a pasta dough, preferably prepared from durum flour ora flour of comparable quality. When used in the preparation of pasta andnoodles, the carbohydrate oxidase may result in a strengthening of thegluten structure and thereby providing a reduction in stickiness of thedough, an increase in dough strength and a dough product with animproved texture.

Baked Product

The process of the invention may be used for any kind of baked productprepared from dough, either of a soft or a crisp character, either of awhite, light or dark type. Examples are bread (in particular white,whole-meal or rye bread), typically in the form of loaves or rolls,French baguette-type bread, pita bread, tortillas, cakes, pancakes,biscuits, cookies, muffins, pie crusts, crisp bread, steamed bread,pizza and the like.

Pre-mix

The present invention further relates to a pre-mix, e.g., in the form ofa flour composition, of dough and/or baked products made from dough, inwhich the pre-mix comprises the carbohydrate oxidase and optionallyother enzymes as specified above. The pre-mix may be prepared by mixingenzyme the relevant enzyme(s) with a suitable carrier, such as flour,starch, a sugar or a salt. The pre-mix may contain other dough-improvingand/or bread-improving additives, e.g. any of the additives, includingenzymes, mentioned above.

Dough and/or Bread-improving Additive

The carbohydrate oxidase may be provided as a dough and/or breadimproving additive in the form of a granulate or agglomerated powder.The dough and/or bread improving additive preferably has a narrowparticle size distribution with more than 95% (by weight) of theparticles in the range from 25 to 500 μm.

Granulates and agglomerated powders may be prepared by conventionalmethods, e.g. by spraying the amylase onto a carrier in a fluid-bedgranulator. The carrier may consist of particulate cores having asuitable particle size. The carrier may be soluble or insoluble, e.g. asalt (such as NaCl or sodium sulfate), a sugar (such as sucrose orlactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits,or soy.

Amino Acid Sequences

The carbohydrate oxidase may be a polypeptide which is produced byMicrodochium nivale CBS 100236, has an amino acid sequence as shown inSEQ ID NO: 2, or is encoded by a gene present in E. coli NRRL B-30034;or it may be an analogue thereof. The analogue may have at least 50%identity, cross-react immunologically, be an allelic variant or afragment having oxidase activity. The carbohydrate oxidase may furtherbe a polypeptide encoded by a nucleic acid sequence which hybridizesunder low stringency conditions with the nucleic acid sequence of SEQ IDNO:1, its complementary strand, or a subsequence thereof of at least 100nucleotides.

The amino acid sequence shown in SEQ ID NO: 2 has less than 20% identityto known sequences. It is 13.6% identical to the amino acid sequence ofa reticuline oxidase precursor from California poppy (GenPept AccessionNo. 2897944) and 17.8% identical to the amino acid sequence of a6-hydroxy-D-nicotine oxidase from Arthrobacter oxidans (GenPeptAccession No.122805).

An amino acid sequence of a polypeptide may be determined using standardmethods for obtaining and sequencing peptides, for example as describedby Findlay and Geisow, Eds., Protein Sequencing--a Practical Approach,1989, IRL Press. A comparison with prior art amino acid sequences hasshown that SEQ ID NO: 2 has only little homology (<20%) to any prior artamino acid sequence.

The polypeptide may be a variant having an amino acid sequence whichdiffers by no more than three amino acids, preferably by no more thantwo amino acids, and more preferably by no more than one amino acid.

The carbohydrate oxidase may comprise at least one partial sequencewhich is the N-terminal amino acid sequence shown at positions 1-24 ofSEQ ID NO: 2 or the internal sequences shown at positions 229-266,249-271, 303-322, 336-347, 383-404, 405-414 and 420-440 of SEQ ID NO: 2.Alternatively, the carbohydrate oxidase may be at least 50% identicalwith at least one of said partial sequences, preferably at least 60%,more preferably at least 70%, even more preferably at least 80%, evenmore preferably at least 90%, and most preferably at least 97%, whichqualitatively retain the activity of the carbohydrate oxidase(hereinafter referred to as "homologous carbohydrate oxidase") andallelic forms and fragments thereof, wherein the fragments retaincarbohydrate oxidase activity.

In a preferred embodiment, the homologous carbohydrate oxidase comprisesan amino acid sequence which differs by five amino acids, preferably byfour amino acids, more preferably by three amino acids, even morepreferably by two amino acids, and most preferably by one amino acidfrom at least one of said partial amino acid sequences. The carbohydrateoxidase may comprise an allelic form or fragment thereof, wherein thefragment retains carbohydrate oxidase activity.

The amino acid sequence of the homologous carbohydrate oxidase maydiffer from any of the partial amino acid sequences by an insertion ordeletion of one or more amino acid residues and/or the substitution ofone or more amino acid residues by different amino acid residues. Theamino acid changes are preferably of a minor nature, that is,conservative amino acid substitutions that do not significantly affectthe tertiary structure and/or activity of the carbohydrate oxidase.Minor amino acid changes may also include small deletions, typically ofone to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a polyhistidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (such as arginine, lysine and histidine), acidic amino acids(such as glutamic acid and aspartic acid), polar amino acids (such asglutamine and asparagine), hydrophobic amino acids (such as leucine,isoleucine and valine), aromatic amino acids (such as phenylalanine,tryptophan and tyrosine) and small amino acids (such as glycine,alanine, serine, and threonine). Amino acid substitutions which do notgenerally alter the specific activity are known in the art and aredescribed, e.g., by H. Neurath and R. L. Hill, 1979, in The Proteins,Academic Press, New York. The most commonly occurring exchanges are:Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val,Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu,Asp/Gly as well as these in reverse.

Nucleic Acid Sequences

The invention provides a nucleic acid sequence comprising a nucleic acidsequence which encodes the carbohydrate oxidase. The carbohydrateoxidase-encoding nucleic acid sequence may comprise:

a) the carbohydrate oxidase encoding part of the DNA sequence clonedinto a plasmid present in Escherichia coli NRRL B-30034, or

b) the DNA sequence shown in positions 67-1550 of SEQ ID NO: 1, or

c) an analogue of the DNA sequence defined in a) or b) which

i) has at least 50% identity with said DNA sequence, or

ii) hybridizes at low stringency with said DNA sequence, itscomplementary strand or a subsequence thereof.

The degree of identity may be at least 60%, preferably about 70%,preferably about 80%, more preferably about 90%, even more preferablyabout 95%, and most preferably about 97%.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

Hybridization indicates that the analogous nucleic acid sequencehybridizes to the oligonucleotide probe under low, medium or highstringency conditions (for example, prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 mg/ml sheared and denatured salmonsperm DNA, and either 50, 35 or 25% formamide for high, medium and lowstringencies, respectively), following standard Southern blottingprocedures. In a preferred embodiment, the nucleic acid sequences arecapable of hybridizing under high stringency conditions with thecarbohydrate oxidase encoding region of at the nucleic acid sequence forthe carbohydrate oxidase of the present invention harbored in CBS100236, its complementary strand, or a subsequence thereof.

The DNA sequence encoding a carbohydrate oxidase may be isolated fromany cell or microorganism producing the carbohydrate oxidase inquestion, using various methods well known in the art to relocate thenucleic acid sequence from its natural location to a different sitewhere it will be reproduced.

The carbohydrate oxidase encoding region of the nucleic acid sequenceharbored in CBS 100236, or subsequences thereof, may be used to designan oligonucleotide probe to isolate homologous genes encodingcarbohydrate oxidases from other strains of different genera or speciesaccording to methods well known in the art. Thus, a genomic or cDNAlibrary prepared from such other organisms may be screened for DNA whichhybridizes with such probes following standard Southern blottingprocedures, in order to identify and isolate the corresponding genetherein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, preferably at least 25, and morepreferably at least 40 nucleotides in length. Longer probes, preferablyno more than 1200 nucleotides in length, can also be used. Both DNA andRNA probes can be used. The probes are typically labeled for detectingthe corresponding gene (for example, with 32P, 3H, biotin, or avidin).According to the present invention, preferred probes may be constructedon the basis of SEQ ID NO: 1.

Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques known in the art. DNA from the libraries or the separated DNAmay be transferred to and immobilized on nitrocellulose or othersuitable carrier material. In order to identify clones or DNA which arehomologous with the nucleic acid sequence for the carbohydrate oxidaseof the present invention harbored in CBS 100236, the carrier material isused in a Southern blot in which the carrier material is finally washedthree times for 30 minutes each using 2×SSC, 0.2% SDS at preferably nothigher than 40° C., more preferably not higher than 45° C., morepreferably not higher than 50° C., more preferably not higher than 55°C., even more preferably not higher than 60° C., especially not higherthan 65° C. Molecules to which the oligonucleotide probe hybridizesunder these conditions are detected using X-ray film.

The isolated nucleic acid sequences of the present invention which arecapable of hybridizing with an oligonucleotide probe which hybridizeswith the nucleic acid sequence for the carbohydrate oxidase of thepresent invention harbored in CBS 100236, its complementary strand, or asubsequence thereof, may be obtained from microorganisms of any genus,for example, from a bacterial or fungal source.

The carbohydrate oxidase may be obtained from (or endogenous to) a givenmicrobial source. Thus, the carbohydrate oxidase may be produced by thesource organism or by a cell in which a gene from the source has beeninserted.

Furthermore, homologous genes may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The nucleic acid sequence may then be derived by similarlyscreening a genomic or cDNA library of another microorganism.

Once a nucleic acid sequence has been detected with the probe(s)described above, the sequence may be isolated or cloned by utilizingtechniques which are well known to those of ordinary skill in the art(see, e.g., Sambrook et al., 1989, supra). The known techniques used toisolate or clone a nucleic acid sequence include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) using specific primers, for instance as described in U.S. Pat. No.4,683,202 or R. K. Saiki et al. (1988, Science 239:487-491). Also see,for example, Innis, et al., 1990, PCR Protocols: A Guide to Methods andApplication, Academic Press, New York. The nucleic acid sequence may becloned from an organism producing the carbohydrate oxidase, or anotheror related organism and thus, for example, may be an allelic or speciesvariant of the carbohydrate oxidase encoding region of the nucleic acidsequence.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoamiditemethod described by S. L. Beaucage and M. H. Caruthers (1981,Tetrahedron Letters 22:1859-1869) or the method described by Matthes etal. (1984, The EMBO J. 3:801-805). In the aforementioned phosphoamiditemethod, oligonucleotides are synthesized, e.g. in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

Modification of the nucleic acid sequence encoding the carbohydrateoxidase may be necessary for the synthesis of a carbohydrate oxidasesubstantially similar to the carbohydrate oxidase. The term"substantially similar" to the carbohydrate oxidase refers tonon-naturally occurring forms of the carbohydrate oxidase. Thiscarbohydrate oxidase may differ in some engineered way from thecarbohydrate oxidase isolated from its native source. For example, itmay be of interest to synthesize variants of the carbohydrate oxidasewhere the variants differ in specific activity, thermostability,oxidative stability, pH optimum, or the like using, for example,site-directed mutagenesis. The analogous sequence may be constructed onthe basis of the carbohydrate oxidase encoding region of the nucleicacid sequence for the carbohydrate oxidase of the present inventionharbored in CBS 100236, a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the carbohydrate oxidase encoded by the nucleic acidsequence, but which corresponds to the codon usage of the host organismintended for production of the enzyme, or by introduction of nucleotidesubstitutions which may give rise to a different amino acid sequence.For a general description of nucleotide substitution, see, e.g., Ford,et al., 1991, Protein Expression and Purification 2:95-107.

Such substitutions can be made outside the regions critical to thefunction of the molecule and still result in an active carbohydrateoxidase. Amino acid residues essential to the activity of thecarbohydrate oxidase encoded by the isolated nucleic acid sequence , andtherefore preferably not subject to substitution, may be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham andWells, 1989, Science 244:1081-1085). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for protease activity toidentify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of crystal structure as determined by such techniques asnuclear magnetic resonance analysis, crystallography or photoaffinitylabeling (see, e.g., de Vos et al., 1992, Science 255:306-312; Smith, etal., 1992, Journal of Molecular Biology 224:899-904; Wlodaver, et al.,1992, FEBS Letters 309:59-64).

The carbohydrate oxidase may be a fused polypeptide in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide or fragment thereof. A fused polypeptide is produced byfusing a nucleic acid sequence (or a portion thereof) encoding anotherpolypeptide to a nucleic acid sequence (or a portion thereof) of thepresent invention. Techniques for producing fusion polypeptides areknown in the art, and include, ligating the coding sequences encodingthe polypeptides so that they are in frame and that expression of thefused polypeptide is under control of the same promoter(s) andterminator.

Yet another method for identifying carbohydrate oxidase-encoding cloneswould involve inserting fragments of genomic DNA into an expressionvector, such as a plasmid, transforming carbohydrate oxidase-negativebacteria with the resulting genomic DNA library, and then plating thetransformed bacteria onto agar containing a substrate for carbohydrateoxidase, thereby allowing clones expressing carbohydrate oxidase to beidentified.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared in accordance with standard techniques by ligating fragments ofsynthetic, genomic or cDNA origin as appropriate wherein the fragmentscorrespond to various sections of the entire DNA sequence.

Identity of Amino Acid or Nucleic Acid Sequences

The polypeptide identity referred to in this specification with claimsis determined as the degree of identity between two sequences indicatinga derivation of the first sequence from the second. The identity maysuitably be determined according to the method described in Needleman,S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48,443-45, with the following settings for polypeptide sequence comparison:GAP creation penalty of 3.0 and GAP extension penalty of 0.1. Thedetermination may be done by means of a computer program known such asGAP provided in the GCG program package (Program Manual for theWisconsin Package, Version August 8, 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711).

Alternatively, the degree of identity may be determined by the Clustalmethod (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10, and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5.

The mature region of an analogous polypeptide may exhibit a degree ofidentity preferably of at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, and especially atleast 95% with the sequence of the carbohydrate oxidase described above.

For purposes of the present invention, the degree of identity betweentwo nucleic acid sequences is determined by the Clustal method (Higgins,1989, CABIOS 5: 151-153) with an identity table, a gap penalty of 10,and a gap length penalty of 10.

Immunochemical Properties

The carbohydrate oxidase may have immunochemical identity or partialimmunochemical identity to a carbohydrate oxidase native to a strain ofM. nivale, or a teleomorph thereof, expressing carbohydrate oxidaseactivity. In this embodiment, said carbohydrate oxidase is used toproduce antibodies which are immunoreactive or bind to epitopes of thepolypeptide.

A polypeptide has immunochemical identity to the polypeptide native toM. nivale means if an antiserum containing antibodies against thepolypeptide native to M. nivale reacts with the other polypeptide in anidentical manner, such as total fusion of precipitates, identicalprecipitate morphology, and/or identical electrophoretic mobility usinga specific immunochemical technique. A further explanation ofimmunochemical identity is described by Axelsen, Bock, and Kr.oslashed.ll in N. H. Axelsen, J. Kr.o slashed.ll, and B. Weeks, editors,A Manual of Quantitative Immunoelectrophoresis, Blackwell ScientificPublications, 1973, Chapter 10.

Partial immunochemical identity means that an antiserum containingantibodies against the polypeptide native to M. nivale reacts with theother polypeptide in a partially identical manner, such as partialfusion of precipitates, partially identical precipitate morphology,and/or partially identical electrophoretic mobility using a specificimmunochemical technique. A further explanation of partialimmunochemical identity is described by Bock and Axelsen in N. H.Axelsen, J. Kr.o slashed.ll, and B. Weeks, editors, A Manual ofQuantitative Immunoelectrophoresis, Blackwell Scientific Publications,1973, Chapter 11.

The immunochemical properties may be determined by immunologicalcross-reaction identity tests, such as the well-known Ouchterlony doubleimmunodiffusion procedure. Specifically, an antiserum against thepolypeptide is raised by immunizing rabbits (or rodents) according tothe procedure described by Harboe and Ingild, in A Manual ofQuantitative Immunoelectrophoresis, N. H. Axelsen, J. Kr.o slashed.ll,and B. Weeks, editors, Blackwell Scientific Publications, 1973, Chapter23, or Johnstone and Thorpe in Immunochemistry in Practice, BlackwellScientific Publications, 1982 (more specifically pages 27-31).

Preferably, the antibodies are monoclonal antibodies. Monoclonalantibodies may be prepared, e.g., according to the methods described inAntibodies, A Laboratory Manual, E. Harlow and D. Lane, editors, 1988,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Purifiedimmunoglobulins may be obtained from the antiserum, e.g., by ammoniumsulfate precipitation, followed by dialysis and ion exchangechromatography (e.g., DEAE-Sephadex).

Production of Carbohydrate Oxidase

The carbohydrate oxidase may be produced by fermentation of the abovementioned microbial strain on a nutrient medium containing suitablecarbon and nitrogen sources and inorganic salts, using procedures knownin the art (e.g., Bennett, J. W., and La Sure, L., eds., More GeneManipulations in Fungi, Academic Press, CA, 1991.) Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). A temperature in the range of from 20° C. to 30° C. issuitable for growth and carbohydrate oxidase production.

The fermentation may be any method of cultivation of a cell resulting inthe expression or isolation of said carbohydrate oxidase. Fermentationmay therefore be understood as comprising shake flask cultivation, smallor large scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thecarbohydrate oxidase to be expressed or isolated.

The resulting carbohydrate oxidase produced by the methods describedabove may be recovered from the fermentation medium by conventionalprocedures including, but not limited to, centrifugation, filtration,spray-drying, evaporation, or precipitation. The recovered protein maythen be further purified by a variety of chromatographic procedures,e.g., ion exchange chromatography, gel filtration chromatography,affinity chromatography, or the like.

The carbohydrate oxidase may be produced by a method comprising (a)cultivating an organism, which in its wild-type form expresses thecarbohydrate oxidase, to produce a supernatant comprising thecarbohydrate oxidase; and (b) recovering the carbohydrate oxidase.

Alternatively, the carbohydrate oxidase may be produced by aerobiccultivation of a transformed host organism containing the appropriategenetic information from the above mentioned strain. Such transformantscan be prepared and cultivated by methods known in the art as describedbelow.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences capable of directing the expression of thecoding sequence in a suitable host cell under conditions compatible withthe control sequences.

The nucleic acid construct may be a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or which has been modified to contain segments of nucleic acidwhich are combined and juxtaposed in a manner which would not otherwiseexist in nature. The nucleic acid construct may be an expressioncassette when the nucleic acid construct contains all the controlsequences required for expression of a coding sequence of the presentinvention. The coding sequence may be a sequence which is transcribedinto mRNA and translated into a carbohydrate oxidase of the presentinvention when placed under the control of the appropriate controlsequences. The boundaries of the coding sequence are generallydetermined by a translation start codon ATG at the 5'-terminus and atranslation stop codon at the 3'-terminus. A coding sequence caninclude, but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

An isolated nucleic acid sequence of the present invention may bemanipulated in a variety of ways to provide for expression of thecarbohydrate oxidase. Manipulation of the nucleic acid sequence prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying nucleic acidsequences utilizing cloning methods are well known in the art.

The control sequences may include all components which are necessary oradvantageous for expression of the coding sequence of the nucleic acidsequence. Each control sequence may be native or foreign to the nucleicacid sequence encoding the carbohydrate oxidase. Such control sequencesinclude, but are not limited to, a leader, a promoter, a signalsequence, and a transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a carbohydrate oxidase.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by the host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptioncontrol sequences which mediate the expression of the carbohydrateoxidase. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice and may be obtainedfrom genes encoding extracellular or intracellular carbohydrate oxidaseeither homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host, are the promoters obtained from the E. coli lac operon,the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilislevansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene(amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM),the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacilluslicheniformis penicillinase gene (penP), the Bacillus subtilis xylA andxylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff etal., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731), as well as the tac promoter i (DeBoer et al., 1983,Proceedings of the National Academy of Sciences USA 80: 21-25). Furtherpromoters are described in "Useful proteins from recombinant bacteria"in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989,supra.

For transcription in a fungal host, examples of useful promoters includethose derivable from the gene encoding the Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutrala-amylase, A. niger acid stable a-amylase, A. niger glucoamylase,Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triosephosphate isomerase and A. nidulans acetamidase.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by the host cell of choice to terminatetranscription. The terminator sequence is operably linked to the 3'terminus of the nucleic acid sequence encoding the carbohydrate oxidase.Any terminator which is functional in the host cell of choice may beused in the present invention.

The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA which is important for translation by thehost cell. The leader sequence is operably linked to the 5' terminus ofthe nucleic acid sequence encoding the carbohydrate oxidase. Any leadersequence which is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of thecarbohydrate oxidase which can direct the expressed carbohydrate oxidaseinto the cell's secretory pathway. The signal peptide coding region maybe native to the carbohydrate oxidase or may be obtained from foreignsources. The 5' end of the coding sequence of the nucleic acid sequencemay inherently contain a signal peptide coding region naturally linkedin translation reading frame with the segment of the coding region whichencodes the secreted carbohydrate oxidase. Alternatively, the 5' end ofthe coding sequence may contain a signal peptide coding region which isforeign to that portion of the coding sequence which encodes thesecreted carbohydrate oxidase. The foreign signal peptide coding regionmay be required where the coding sequence does not normally contain asignal peptide coding region. Alternatively, the foreign signal peptidecoding region may simply replace the natural signal peptide codingregion in order to obtain enhanced secretion of the carbohydrate oxidaserelative to the natural signal peptide coding region normally associatedwith the coding sequence. Any signal peptide coding region capable ofdirecting the expressed carbohydrate oxidase into the secretory pathwayof the host cell of choice may be used in the present invention.

An effective signal peptide coding region for a bacterial host cell, inparticular, Bacillus, is the signal peptide coding region obtained fromthe maltogenic amylase gene from Bacillus NCIB 11837, the Bacillusstearothermophilus alpha-amylase gene, the Bacillus licheniformissubtilisin gene, the Bacillus licheniformis beta-lactamase gene, theBacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM),and the Bacillus subtilis prsA gene. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the carbohydrate oxidase at suchsites. Alternatively, the nucleic acid sequence of the present inventionmay be expressed by inserting the nucleic acid sequence or a nucleicacid construct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression, and possiblysecretion.

The expression vector may also comprise in eukaryotes a poly-adenylationsequences operably linked to the DNA sequence encoding the carbohydrateoxidase. Termination and poly-adenylation sequences may be suitablyderived from the same sources as the promoter.

The recombinant expression vector may be any vector which can beconveniently subjected to recombinant DNA procedures and can bring aboutthe expression of the nucleic acid sequence. The choice of the vectorwill typically depend on the compatibility of the vector with the hostcell into which the vector is to be introduced. The vectors may belinear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i.e., a vector which exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone which, when introduced into the host cell, is integrated into thegenome and replicated together with the chromosome(s) into which it hasbeen integrated. The vector system may be a single vector or plasmid,two or more vectors or plasmids which together contain the total DNA tobe introduced into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocideresistance, resistance to heavy metals, prototrophy to auxotrophs, andthe like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,erythromycin, chloramphenicol or tetracycline resistance. Furthermore,selection may be accomplished by cotransformation, e.g., as described inWO 91/09129, where the selectable marker is on a separate vector.

The vectors of the present invention contain an element(s) that permitsstable integration of the vector into the host cell genome or autonomousreplication of the vector in the cell independent of the genome of thecell.

The vectors of the present invention may be integrated into the hostcell genome when introduced into a host cell. For integration, thevector may rely on the nucleic acid sequence encoding the carbohydrateoxidase or any other element of the vector for stable integration of thevector into the genome by homologous recombination. Alternatively, thevector may contain additional nucleic acid sequences for directingintegration by homologous recombination into the genome of the hostcell. The additional nucleic acid sequences enable the vector to beintegrated into the host cell genome at a precise location in thechromosome. To increase the likelihood of integration at a preciselocation, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 1,500 base pairs,preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500base pairs, which are highly homologous with the corresponding targetsequence to enhance the probability of homologous recombination. Theintegrational elements may be any sequence that is homologous with thetarget sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleic acidsequences.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. The origin of replication may be onehaving a mutation to make its function temperature-sensitive in theBacillus cell (see, e.g., Ehrlich, 1978, Proceedings of the NationalAcademy of Sciences USA 75:1433).

More than one copy of a nucleic acid sequence encoding a carbohydrateoxidase of the present invention may be inserted into the host cell toamplify expression of the nucleic acid sequence. Stable amplification ofthe nucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome using methodswell known in the art and selecting for transformants. A convenientmethod for achieving amplification of genomic DNA sequences is describedin WO 94/14968.

Procedures suitable for constructing vectors encoding a carbohydrateoxidase and containing the promoter, terminator and other elements,respectively, are well known to persons skilled in the art (c.f., forinstance, Sambrook et al., supra).

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the carbohydrate oxidase relative to thegrowth of the host cell. Examples of regulatory systems are those whichcause the expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems would include thelac, tac, and trp operator systems. Other examples of regulatorysequences are those which allow for gene amplification. In these cases,the nucleic acid sequence encoding the carbohydrate oxidase would beoperably linked with the regulatory sequence.

While intracellular expression may be advantageous in some respects,e.g., when using certain bacteria as host cells, it is generallypreferred that the expressed carbohydrate oxidase is secretedextracellularly.

Host Cells

The present invention also relates to recombinant host cells, eithercomprising a DNA construct or an expression vector as described above,which are advantageously used in the recombinant production of thecarbohydrate oxidase. The host cell may be any progeny of a parent cellwhich is not identical to the parent cell due to mutations that occurduring replication.

The cell is preferably transformed with a vector comprising a nucleicacid sequence followed by integration of the vector into the hostchromosome. "Transformation" means introducing a vector comprising anucleic acid sequence of the present invention into a host cell so thatthe vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector. Integration is generallyconsidered to be an advantage as the nucleic acid sequence is morelikely to be stably maintained in the cell. Integration of the vectorinto the host chromosome occurs by homologous or non-homologousrecombination as described above.

The choice of a host cell will to a large extent depend upon the geneencoding the carbohydrate oxidase and its source. The host cell may be acell of a higher organism, such as a mammal or an insect, but ispreferably a microbial cell, e.g., a bacterial or a fungal (includingyeast) cell.

Examples of suitable bacterial cells are gram positive bacteriaincluding, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans orStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus orBacillus subtilis cell.

The transformation of a bacterial host cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168:111-115), by using competent cells (see,e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169:5771-5278).

The host cell may also be a eukaryote, such as a mammalian cell, aninsect cell, a plant cell or a fungal cell. Useful mammalian cellsinclude Chinese hamster ovary (CHO) cells, HeLa cells, baby hamsterkidney (BHK) cells, COS cells, or any number of other immortalized celllines available, e.g., from the American Type Culture Collection.

In a preferred embodiment the host cell is a fungal cell. "Fungi," asused herein, includes the phyla Ascomycota, Basidomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth, et al.,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth, et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth, et al., 1995, supra). Representativegroups of Ascomycota include, e.g., Neurospora, Eupenicillium(=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), andthe true yeasts listed above. Examples of Basidiomycota includemushrooms, rusts, and smuts. Representative groups of Chytridiomycotainclude, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquaticfungi. Representative groups of Oomycota include, e.g.,Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examplesof mitosporic fungi include Aspergillus, Penicillium, Candida, andAlternaria. Representative groups of Zygomycota include, e.g., Rhizopusand Mucor.

In a preferred embodiment, the fungal host cell is a yeast cell. "Yeast"as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). The ascosporogenous yeasts are divided into thefamilies Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae, andSaccharomycoideae (e.g., genera Pichia, Kluyveromyces andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeast belonging to the Fungi Imperfecti are divided intotwo families, Sporobolomycetaceae (e.g., genera Sorobolomyces andBullera) and Cryptococcaceae 6 (e.g., genus Candida). The yeast may beas described in Biology and Activities of Yeast (Skinner, F. A.,Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol.Symposium Series No. 9, 1980. The biology of yeast and manipulation ofyeast genetics are well known in the art (see, e.g., Biochemistry andGenetics of Yeast, Bacil, M., Horecker, B. J., and Stopani, A. O. M.,editors, 2nd edition, 1987; The Yeasts, Rose, A. H., and Harrison, J.S., editors, 2nd edition, 1987; and The Molecular Biology of the YeastSaccharomyces, Strathern et al., editors, 1981).

In a more preferred embodiment, the yeast host cell is a cell of aspecies of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces,Pichia, or Yarrowia.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In a preferred embodiment, the fungal host cell is a filamentous fungalcell. "Filamentous fungi" include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a vegetativemycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative. In a more preferred embodiment, the filamentous fungalhost cell is a cell of a species of, but not limited to, Acremonium,Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora,Penicillium, Thielavia, Tolypocladium, and Trichoderma.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium cerealis, Fusarium crookwellense, Fusarium graminearum,Fusarium oxysporum, Fusarium sambucinum, Fusarium sulphureum, orFusarium venenatum cell. In another most preferred embodiment, thefilamentous fungal host cell is a Humicola insolens or Humicolalanuginosa cell. In another most preferred embodiment, the filamentousfungal host cell is a Mucor miehei cell. In another most preferredembodiment, the filamentous fungal host cell is a Myceliophthorathermophilum cell. In another most preferred embodiment, the filamentousfungal host cell is a Neurospora crassa cell. In another most preferredembodiment, the filamentous fungal host cell is a Penicilliumpurpurogenum cell. In another most preferred embodiment, the filamentousfungal host cell is a Thielavia terrestris cell. In another mostpreferred embodiment, the Trichoderma cell is a Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei orTrichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81:1470-1474. A suitable method of transforming Fusarium species isdescribed by Malardier et al., 1989, Gene 78:147-156 or in copendingU.S. Ser. No. 08/269,449. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, in Abelson, J. N. and Simon, M. I.,editors, "Guide to Yeast Genetics and Molecular Biology", Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito,et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75:1920. Mammaliancells may be transformed by direct uptake using the calcium phosphateprecipitation method of Graham and Van der Eb (1978, Virology 52:546).

Recombinant Methods of Production

The carbohydrate oxidase can be produced by recombinant methodscomprising cultivating a host cell as described above under conditionsconducive to the production of said carbohydrate oxidase and recoveringthe carbohydrate oxidase from the cells and/or culture medium.

In these methods, the cells are cultivated in a nutrient medium suitablefor production of the carbohydrate oxidase using methods known in theart. For example, the cell may be cultivated by shake flask cultivation,small-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe carbohydrate oxidase to be expressed and/or isolated. Thecultivation takes place in a suitable nutrient medium comprising carbonand nitrogen sources and inorganic salts, using procedures known in theart (see, e.g., M. V. Arbige et al., in Abraham L. Sonenshein, James A.Hoch, and Richard Losick, editors, Bacillus subtilis and OtherGram-Positive Bacteria, American Society For Microbiology, Washington,D.C., 1993, pages 871-895). Suitable media are available from commercialsuppliers or may be prepared ac-cording to published compositions (e.g.,in catalogues of the American Type Culture Collection). If thecarbohydrate oxidase is secreted into the nutrient medium, thecarbohydrate oxidase can be recovered directly from the medium. If thecarbohydrate oxidase is not secreted, it is re-covered from celllysates.

The carbohydrate oxidase may be detected using methods known in the artthat are specific for the polypeptide. These detection methods mayinclude use of specific anti-bodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the polypeptide.

The resulting carbohydrate oxidase may be recovered by methods known inthe art. For example, the carbohydrate oxidase may be recovered from thenutrient medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray-drying, evaporation,or precipitation.

The carbohydrate oxidase of the present invention may be purified by avariety of procedures known in the art including, but not limited to,chromatography (e.g., ion ex-change, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

Industrial Applications

In addition to the us in baking, discussed above, the carbohydrateoxidase may be used, for example, in personal care products such astoothpaste, in particular, where whitening of the teeth is desirable,mouthwash, denture cleaner, liquid soap, skin care creams and lotions,hair care and body care formulations, and solutions for cleaning contactlenses in an amount effective to act as an antibacterial agent. Thecarbohydrate oxidase may also be a component of a laundry detergentcomposition or a dishwashing detergent composition and may be used forthe generation of hydrogen peroxide. The laundry detergent compositionmay comprise a surfactant, said carbohydrate oxidase and a substrate forthe carbohydrate oxidase. The dishwashing detergent composition maycomprise said carbohydrate oxidase and a bleach precursor or peroxyacid, and a substrate for carbohydrate oxidase.

The carbohydrate oxidase may be used as an analytical reagent, forexample, to determine the amount of reducing sugars present in a givensample, or the enzyme may be immobilized and inserted into an electrodeto provide continuous measurement of starch or cellulose hydrolysis.

In addition, the carbohydrate oxidase of the present invention may beused to oxidize an oligosaccharide with a glucose residue at thereducing end into the corresponding acid, e.g. to produce lactobionicacid from lactose.

Methods for Determination of Carbohydrate Oxidase Activity

DMAB/MBTH Assay

Premix:

7.2 mM 3-dimethylaminobenzoic acid (DMAB)

0.33 mM 3-methyl-2-benzothiazolinone hydrazone (MBTH)

4 mg/ml recombinant Coprinus cinereus peroxidase (rCiP)

0.4M/0.4M Phosphate/Citrate buffer (pH 6)

Incubation mix:

180 μl 500 mM glucose 25 mM citrate 25 mM phosphate pH 6.0

20 μl Sample

The incubation mix is allowed to incubate for 20 minutes at 30° C. Then100 ml of the incubation mix and 100 ml premix are mixed together. After30 seconds, the absorbance at 540 (or 490) nm is read. A standard of 0.2mM H2O2 is included.

4AA-TOPS Assay

Assays are carried out in 96 well microtiter plates. 100 μl 0.1 Mphosphate/citrate, pH 6 is mixed with 50 μl 0.24 M glucose and 50 μlpre-mix (3 mM 4-aminoantipyrine (4AA), 7 mMN-ethyl-N-sulfopropyl-m-toluidine (TOPS), 40 PODU/ml rCiP) and thereaction is started by adding 40 μl of oxidase solution dilutedappropriately. Absorption is measured at 490 nm as a function time usingthe Vmax microtiter plate ready from Molecular Devices and the activityis taken as the slope of the linear increase in absorption.

EXAMPLES Example 1

Production of Carbohydrate Oxidase from Wild-type M. nivale

Cultivation of M. nivale

A strain of M. nivale, CBS 100236, was fermented using the followingcomplete medium:

    ______________________________________                                        Shaking flask medium:           BA                                              Rofec (Roquette) 10 gram                                                      NH.sub.4 NO.sub.3 (Merck) 10 gram                                             KH.sub.2 PO.sub.4 (Merck) 10 gram                                             Solcafloc (Dicacel) 40 gram                                                   MgSO.sub.4 --7(H.sub.2 O) 0.75 gram                                           Pluronic 100% (BASF) 0.1 ml                                                   Tap water for a final volume of 1000 ml                                     ______________________________________                                    

The pH was adjusted to pH 6.5, then 1 tablet of 500 mg CaCO₃ was added.One hundred ml of the complete medium was added to each 500 ml 2-baffleshake flask. The shake flasks were then autoclaved for 40 min. at 121°C. An inoculum was prepared from a spore suspension prepared from 5 PDAslants, grown for 7 days at 26° C., then washed in 20 ml sterile waterand Tween 80 (ICI). Each shake flask was inoculated with 2 ml of thespore suspension, then cultured for 10 days at 26° C. and with constantshaking at 125 rpm. At the end of cultivation, the cells were pelleted,and the enzyme was purified from the supernatant.

Purification

From a 5 liter fermentation, 4300 ml of centrifuged fermentation brothwas filtered and concentrated to 660 ml by ultrafiltration using afilter with a molecular weight cutoff of 10 kDa (Filtron). The enzymewas precipitated with (NH4)₂ SO₄ between 200 and 400 mg/ml. Afterdissolving the precipitate in 25 mM Tris pH 7.5 the sample was washed byultrafiltration until the conductivity was identical to 25 mM Tris pH7.5. The sample was passed over a column of 300 ml Q-Sepharose XL(Pharmacia) equilibrated in the same buffer and the run-throughcollected. After adding (NH4)₂ SO₄ to 100 mg/ml the sample was passedover a HIC column (Toyopearl-butyl 650) (TosoHaas) equilibrated with 25mM Tris pH 7.5; 100 mg (NH4)₂ SO₄ /ml. The run-through was washed with25 mM acetate buffer pH 5.0 and applied to a column of SP-Sepharose(Pharmacia) equilibrated in the same buffer. Bound enzyme was elutedusing a linear salt gradient to 1 M NaCl in 25 mM acetate buffer pH 5.0over 10 column volumes. Active fractions were pooled. Final polishing ofthe preparation was done by HIC chromatography on a phenyl-superosecolumn, using a buffer of 25 mM acetate buffer pH 5.0 with a lineargradient running from 2 M (NH4)₂ SO₄ to 0 M over 20 column volumes.Active fractions were pooled and dialyzed against 25 mM acetate bufferpH 5.0 for 24 hours.

Characterization

Analysis of the purified protein by SDS-PAGE indicated a molecularweight of approximately 52 kDa and a pl of around 8.9 by isoelectricfocusing.

The purified M. nivale oxidase also showed a pronounced yellow color,suggesting the presence of FAD as a cofactor. An absorbance scan of theenzyme revealed two absorption maxima, at 385 and 440 nm, characteristicof the presence of FAD in the enzyme. In the presence of glucose, thepeak at 440 nm disappeared, indicating a reduction of the FAD.

Example 2

Amino Acid Sequences from M. nivale Carbohydrate Oxidase

A highly purified preparation of M. nivale was reduced and alkylated. Asample of the enzyme was then degraded with Lysyl-endopeptidase (Wako)or TPCK-trypsin (Promega). Peptides were isolated by RP-HPLC on a Vydac218TP column (Vydac) in TFA (trifluoroacetate)/isopropanol andrepurified on a Vydac 218TP column in TFA/acetonitrile. Selectedpeptides were analyzed by Edman degradation. The N-terminal sequence wasdetermined by sequencing the purified enzyme electroblotted onto a PVDFmembrane.

The partial sequences obtained were an N-terminal sequence shown atpositions 1-24 of SEQ ID NO: 2 and internal sequences as shown atpositions 229-266, 249-271, 303-322, 336-347, 383-404, 405-414, 420-440of SEQ ID NO: 2. None of the sequences from the carbohydrate oxidaseshowed homology to any relevant sequences when searched against theSwissprot and EMBL databases.

Example 3

Extraction of Microdochium nivale Genomic DNA

Agar slants of Microdochium nivale (NN008551, CBS 100236) mycelia wererinsed with 10 ml of sterile 0.008% Tween 20. A 2 ml volume of themycelial solution was inoculated into a 250 ml shake flask containing 50ml of MY50 pH 6.0 medium. The MY50 pH 6.0 medium was composed per literof 50 g of maltodextrin, 2 g of MgSO₄.7H₂ O, 10 g of KH₂ PO₄, 2 g of K₂SO₄, 2 g of citric acid, 10 g of yeast extract, 2 g of urea, and 0.5 mlof AMG trace elements. The AMG trace metals solution was composed perliter of 14.3 g of ZnSO₄.7H₂ O, 2.5 g of CuSO₄.5H₂ O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂ O, 8.5 g of MnSO₄.H₂ O, and 3 g of citric acid.The shake flask was incubated at 26° C., 125 rpm for 6 days.

Mycelia from the 6 day culture were collected through Miracloth(Calbiochem, La Jolla, Calif.), rinsed twice with approximately 50 ml of10 mM Tris-1 mM EDTA pH 8.0 (TE), and squeezed dry. The mycelia werethen frozen in liquid nitrogen and ground to a fine powder in anelectric coffee grinder pre-chilled with dry ice. A 2 g sample of thepowder was transferred to a sterile disposable 50 ml conical tube and a20 ml volume of lysis buffer (100 mM EDTA, 10 mM Tris, 1% Triton X-100,500 mM guanidine-HCl, 200 mM NaCl, pH 8.0) was added slowly followed by20 μg of DNase-free RNase A per ml. The mixture was incubated at 37° C.for 30 minutes. Proteinase K was then added at 0.8 mg per ml and themixture was incubated at 50° C. for an additional 2 hours. The lysedmixture was centrifuged at 12-15,000×g for 20 minutes to pellet theinsoluble debris.

The lysate supernatant was transferred to a Qiagen-tip 500 Maxi column(Qiagen, Santa Clarita, Calif.) pre-equilibrated with 10 ml of QBTbuffer (Qiagen, Santa Clarita, Calif.) and the column was washed with 30ml of QC buffer (Qiagen, Santa Clarita, Calif.). The DNA was eluted with15 ml QF buffer (Qiagen, Santa Clarita, Calif.) and 7 volumes of filtersterilized isopropanol was added to the eluted DNA solution. Thesolution was mixed gently and then centrifuged for 20 minutes at15,000×g to pellet the DNA. The pelleted DNA was washed with ml ofice-cold 70% ethanol, air dried, and re-suspended in 500 μl of TE.

Example 4

PCR Amplification of Microdochium nivale Carbohydrate Oxidase Gene

The primary amino acid sequence data from the N-terminal and internalfragments of the purified Microdochium nivale carbohydrate oxidasedescribed in Example 2 was used to create the following degenerate PCRprimers to amplify the carbohydrate oxidase gene from the genomicMicrodochium nivale DNA prepared in Example 3:

    Forward primer (N-terminal peptide sequence): GCIGCIGGIGTICCIATHGAYAT(SEQ ID NO:3)

    Reverse Primer (internal peptide sequence): IGGRTCIGCRTARTTDATRTACAT(SEQ ID NO:4)

Amplification was accomplished using the Hot Wax Optistart™ Kit(Invitrogen, San Diego, Calif.) according to the manufacturer'sinstructions. Six reactions were set up that differ from each other bypH and Mg²⁺ concentration as shown below in the following table:

    ______________________________________                                        1.5 mM MgCl.sub.2                                                                              2.5 mM MgCl.sub.2                                                                        3.5 mM MgCl.sub.2                                 ______________________________________                                        pH 8.5  reaction 1   reaction 2 reaction 3                                      pH 9  reaction 4                                                              pH 9.5  reaction 5                                                            pH 10  reaction 6                                                           ______________________________________                                    

The amplification reactions (50 μl) contained 1.62 μg of Microdochiumnivale genomic DNA as the template, 50 pmole of each primer, 1×PCRBuffer, 5 μl of a 10 mM dNTP mix, and a HotWax™ Mg²⁺ bead containing theappropriate amount of Mg²⁺. The reactions were cycled in a Perkin Elmer480 Thermal Cycler programmed as follows: Cycle 1 at 94° C. for 2.5minutes and 72° C. for 2 minutes; cycles 2-37 each at 94° C. for 45seconds, 50° C. for 45 seconds, and 72° C. for 2 minutes; and cycle 38 a94° C. for 45 seconds, 50° C. for 45 seconds, and 72° C. for 10 minutes.Cycle 39 was a 4° C. soak cycle.

A 9 μl volume from each reaction was electrophoresed on a 1% agarose gelusing 50 mM Tris-50 mM boric acid-1 mM EDTA (TBE) buffer. Reactions 4,5, and 6 revealed a major band at 1335 bp. Reactions 4, 5, and 6, werepooled and electrophoresed on a 1% agarose gel as before. The 1335 bpband was excised from the gel and purified using a Qiaex II GelExtraction Kit (Qiagen, Santa Clarita, Calif.). The purified 1335 bp PCRproduct was then cloned into pCR2.1-TOPO (Invitrogen, San Diego, Calif.)and transformed into Escherichia coli TOP10 cells (Invitrogen, Carlsbad,Calif.) according to the manufacturer's instructions. Plasmid DNA wasisolated from the transformants using a Wizard Maxi Prep Kit (Promega,Madison, Wis.). The isolated plasmid DNA was sequenced using an AppliedBiosystems Prism 377 DNA Sequencer and 377XL collection and analysissoftware (Perkin Elmer, Applied Biosystems, Foster City, Calif.)according to the manufacturer's instructions. The sequence dataconfirmed that the 1335 bp fragment encoded part of the Microdochiumnivale carbohydrate oxidase gene.

Example 5

Southern Blot of Microdochium nivale Genomic DNA

A sample of the genomic DNA prepared in Example 3 was analyzed bySouthern hybridization (Maniatis et al., 1982, Molecular Cloning, aLaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).Approximately 3 μg of the genomic DNA were digested with EcoRI, KpnI,NotI, SacI, SphI, or XbaI (Boehringer Mannheim, Indianapolis, Ind.) andfractionated by size on a 0.6% agarose gel using TBE buffer. The gel wasphotographed under short wavelength UV light and soaked for 30 minutesin 0.5 M NaOH-1.5 M NaCl followed by 15 minutes in 1 M Tris-HCl pH 8-1.5M NaCl. DNA in the gel was transferred onto a Hybond N hybridizationmembrane (Amersham Life Science, Arlington Heights, Ill.) by capillaryblotting in 20×SSPE (3 M sodium chloride-0.2 M sodium dibasicphosphate-0.02 M disodium EDTA) using the Turbo Blot method (Schleicherand Schuell, Keene, N.H.). The membrane was crosslinked with UV (UVStratalinker 2400, Stratagene, La Jolla, Calif.) and then soaked for 2hours in the following hybridization buffer at 45° C. with gentleagitation: 5×SSPE, 50% formamide (v/v), 0.3% SDS, and 200 μg/mldenatured and sheared salmon testes DNA.

The 1335 bp fragment described in Example 4, containing part of thecoding sequence of the Microdochium nivale carbohydrate oxidase, wasradiolabeled by random priming (Prime it II, Stratagene, La Jolla,Calif.) with α[³² P]dCTP (Amersham, Arlington Heights, Ill.). The α[³²P]dCTP-labeled fragment was added to the hybridization buffer at anactivity of approximately 1×10⁶ cpm per ml of buffer. The mixture wasincubated with the membrane overnight at 45° C. in a shaking water bath.Following incubation, the membrane was washed three times for fifteenminutes each in 2×SSC (0.3 M NaCl, 30 mM sodium citrate pH 7.0) with0.2% SDS at 45° C. The membrane was dried on a paper towel for 15minutes, then wrapped in plastic wrap, and exposed to X-ray film forthree hours at -70° C. with intensifying screens (Kodak, Rochester,N.Y.).

The Southern blot showed the presence of a 3 kb band in the lanecontaining the SacI digest. Since the SacI digest yielded a band sizesmall enough to amplify and large enough to contain the full-lengthgene, it was used for isolating the complete gene by inverse PCR.

Example 6

Inverse PCR of Coding Sequence of the Microdochium nivale CarbohydrateOxidase Gene

Inverse PCR was used to obtain the 5' and 3' flanking DNA of the 1335 bpfragment to isolate the entire coding sequence of the Microdochiumnivale carbohydrate oxidase gene. A 6 μg sample of Microdochium nivalegenomic DNA (Example 3) was digested to completion with SacI and thenpurified using the QIAquick Nucleotide Removal Kit (Qiagen, SantaClarita, Calif.) according to the manufacturer's instructions. A 1 μgsample of the purified digested DNA was self-ligated overnight at 14-16°C. with 10 units of T4 Ligase (Boehringer Mannheim, Indianapolis, Ind.)and 1×ligase buffer in a final volume of 500 μl. The ligase was thenheat inactivated by incubating the reaction at 65° C. for 15 minutes.The reaction was concentrated using a Microcon 30 (Millipore, Bedford,Mass.). The reaction products were purified using the QIAquickNucleotide Removal Kit. The self-ligated products were then used as atemplate for inverse PCR.

The following primers were created to the 5' and 3' ends of thecarbohydrate oxidase gene, in the opposite direction to those inconventional PCR, to amplify out from the known region of the 1335 bpPCR product:

    Upper 1199 bp: TCCAGTTCTACGACCGCTACG                       (SEQ ID NO:5)

    Lower 158 bp: CAGACTTGGCAGAGACCTTGA                        (SEQ ID NO:6)

The amplification reaction (100 μl) contained 100 pmoles of each primer,1 μg of the SacI digested and self-ligated genomic DNA, 10 μl of a 10 mMdNTP mix, 1×Taq polymerase buffer (Perkin Elmer, Foster City, Calif.),and 5 units of Taq polymerase (Perkin Elmer, Foster City, Calif.).Sterile mineral oil was layered on top of the reaction and placed in aPerkin Elmer Model 480 Thermal Cycler programmed as follows: Cycle 1 at94° C. for 2.5 minutes and 72° C. for 2 minutes; cycles 2-11 each at 94°C. for 30 seconds, 55° C. for 45 seconds, and 72° C. for 2 minutes;cycles 12-28 each at 94° C. for 30 seconds, 55° C. for 45 seconds, and72° C. for 2 minutes with an extension of 20 seconds per cycle; andcycle 29 at 72° C. for 10 minutes. Cycle 30 was a 4° C. soak cycle.

The reaction was electrophoresed on a 1% agarose gel using TBE bufferrevealing a 3 kb band. The 3 kb band was excised from the gel andpurified using a Qiaex II Gel Extraction Kit. The purified 3 kb PCRproduct was then cloned into pCR2.1-TOPO and transformed intoEscherichia coli TOP10 cells to generate Eschelichla coli pEJG40/TOP10.The transformant E. coli pEJG40/TOP10 was deposited according to theBudapest Treaty on the International Recognition of the Deposits ofMicroorganisms for the Purpose of Patent Procedures on Jun. 12, 1998 atthe Agricultural Research Service Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill., and designated NRRL B-30034.

Plasmid DNA was isolated from the transformant using a Wizard Maxi PrepKit. The isolated plasmid DNA was sequenced using an Applied BiosystemsPrism 377 DNA Sequencer and 377XL collection and analysis softwareaccording to the primer walking technique with dye-terminator chemistry(Giesecke et al., 1992, Journal of Virol. Methods 38: 47-60). Inaddition to the lac-forward and lac-reverse primers, the followingoligonucleotide sequencing primers were used for sequencing:

Sequencing Primers for 1335 bp fragment:

    IACRTCRMRTARTARTCIACRAARTT                                 (SEQ ID NO:7)

    RTTIACCCAICCRTC                                            (SEQ ID NO:8)

    IGGRTCIGCRTARTTDATRTACAT                                   (SEQ ID NO:9)

    DATRAARTCIACRTGRTCRAARTT                                   (SEQ ID NO:10)

    CCAYTGYTCIGGIGTICCRTARTA                                   (SEQ ID NO:11)

    CTCGCCACTTTCCCTGCTCCC                                      (SEQ ID NO:12)

    CTCGGTCACCAAGGCTCTCCC                                      (SEQ ID NO:13)

    GACCGCTACGACMCMCCAG                                        (SEQ ID NO:14)

Sequencing primers for Inverse PCR product:

    TCGGAGAAATGAGAGCMCCA                                       (SEQ ID NO:15)

    AGCCGACGTCCAGCATAGCAG                                      (SEQ ID NO:16)

    ACCCTACCATACGAGTTCACG                                      (SEQ ID NO:17)

    GGTCGAATCGTCACAAAGTAT                                      (SEQ ID NO:18)

    CACTGGACTGCCGACTGGATG                                      (SEQ ID NO:19)

    CACMCMCCAGACCTACCC                                         (SEQ ID NO:20)

    CTCAGCAGCACTTCTTTTCAT                                      (SEQ ID NO:21)

Sequencing revealed a nucleic acid sequence with an open reading frame(ORF) of 1448 bp (SEQ ID NO:1) containing one intron of 65 bp. The G+Ccontent of this ORF is 58.33%. The -3 position of the translationalstart is an adenine agreeing with Kozak's rules in which the -3 positionis always an adenine. A putative TATA motif is also present at -122,TATAAA.

The deduced amino acid sequence (SEQ ID NO:2) demonstrated a protein of495 amino acids with a calculated molecular weight of 54,678 daltons.Based on the rules of van Heijne (van Heijne, 1984, Journal of MolecularBiology 173: 243-251), the first 18 amino acids likely comprise asecretory signal peptide which directs the nascent polypeptide into theendoplasmic reticulum. A score of 30.395 was obtained using the vanHeijne program to predict signal peptides. The amino acid sequences ofthe partial peptides derived from the purified Microdochium nivalecarbohydrate oxidase fragments (Example 2) were consistent with thosefound in the deduced amino acid sequence except there may be a fouramino acid propeptide in that the N-terminal amino acid sequence doesnot follow immediately after the signal peptide.

Example 7

Construction of Microdochium nivale Carbohydrate Oxidase ExpressionVectors

Two synthetic oligonucleotide primers shown below were synthesized toPCR amplify the carbohydrate oxidase gene from Microdochium nivalegenomic DNA for subcloning and expression in Fusarium and Aspergillushost cells. In order to facilitate the subcloning of the gene fragmentinto the expression vectors pDM181 and pBANE15, SwaI and PacIrestriction enzyme sites were introduced at the 5' and 3' end of thecarboxypeptidase gene, respectively.

    Forward primer: 5'-GATTTAAATATGCGTTCTGCATTTATCTTG-3'       (SEQ ID NO:22)

    Reverse primer: 5'-GTTAATTMTTATTTGACAGGGCGGACAGC-3'        (SEQ ID NO:23)

Bold letters (at positions 10-30 of each) represent coding sequence.

The PCR, purification, and subcloning were performed as described inExample 4 except the cycling parameters varied as follows: Cycle 1 at94° C. for 2 minutes, 60° C. for 4 seconds, and 72° C. for 45 seconds;cycles 2-37 each at 94° C. for 45 seconds, 60° C. for 45 seconds, and72° C. for 2 minutes; and cycle 38 at 94° C. for 45 seconds, 60° C. for45 seconds, and 72° C. for 6 minutes. Cycle 39 was a 4° C. soak cycle.

The vector pDM181 contains the Fusarium oxysporum trypsin-like proteasepromoter and terminator as regulatory sequences and the Streptomyceshygroscopicus bar gene as a selectable marker for fungal transformations(WO 98/20136; de Block et al., 1987, EMBO Journal 6: 2513-2518). Thevector pBANE15 (FIG. 1) contains the TAKA promoter and AMG terminatorand the Aspergillus nidulans amdS gene as the selectable marker forfungal transformations. Both vectors also contain the amp gene forselection in E. coli.

The carbohydrate oxidase clone obtained above was digested with SwaI andPacI and purified by 0.8% agarose gel electrophoresis using TBE bufferand a Qiaex II Gel Extraction Kit. The digested fragment was cloned intopDM181 and pBANE15 previously digested with SwaI and PacI resulting inthe expression plasmids pEJG35 and pEJG33, respectively (FIGS. 2 and 3).

The expression plasmids were transformed into E. coli XL10 Gold cells(Stratagene, La Jolla, Calif.). Transformants containing the correctplasmids were isolated and plasmid DNA was prepared using the WizardMaxi Prep Kit.

Example 8

Expression of the Microdochium nivale Carbohydrate Oxidase Gene inAspergillus oryzae

pEJG33 was transformed into protease-deficient Aspergillus oryzae hoststrains JaL142 (Christensen et al., 1988, Bio/Technology 6: 1419-1422)and JaL228 (WO 98/12300) using protoplast transformation (Yelton et al.,1984, Proceedings of the National Academy of Sciences USA 81:1470-1474). One hundred ill of protoplasts (2×10⁶) were placed into a 14ml Falcon tube with ca. 5 μg of pEJG33 and gently mixed. A 250 μl volumeof 60% PEG 4000 in 10 mM Tris-HCl pH 7.5-10 mM CaCl₂ was added and mixedby gentle rolling. The tube was then incubated at 37° C. for 30 minutes.Three ml of STC (1.2 M sorbitol, 10 mM Tris pH 7.5, 10 mM CaCl₂) wasadded and mixed by inversion. The solution was then plated directly ontoCove plates composed per liter of 342.3 g of sucrose, 10 ml of 1.5 MCsCl, 10 ml of 1 M acetamide, 20 ml of 1×Cove salt solution, and 1%agar. 5×Cove salts solution was composed per liter of 26 g of KCl, 26 gof MgSO₄.7H₂ O, 76 g of KH₂ PO₄, and 50 ml of Cove trace metals. TheCove trace metals solution was composed per liter of 0.04 g of NaB₄O₇.10H₂ O, 0.4 g of CuSO₄.5H₂ O, 1.2 g of FeSO₄.7H₂ O, 0.7 g of MnSO₄.H₂O, 0.8 g of Na₂ MoO₂.2H₂ O, and 10 g of ZnSO₄.7H₂ O. Plates wereincubated 5 days at 37° C. Transformants were transferred to plates ofthe same medium and incubated 5 days at 37° C. The transformants werepurified by streaking spores and picking isolated colonies using thesame plates under the same conditions. Totally, 12 Aspergillus oryzaeJaL142 transformants and 22 Aspergillus oryzae JaL228 transformants wererecovered.

The transformants were grown on individual COVE plates as above, andthen tested for carbohydrate oxidase activity using an indicator platedescribed by WiHeveen et al. (1990, Applied Microbial Biotechnology 33:683). The untransformed hosts were used as controls. A total of 11positive transformants were identified. Spore stocks of each positivetransformant were made with sterile deionized water. A 500 μl volume ofeach spore stock including the untransformed host was inoculated into125 ml shake flasks containing 25 ml of MY50 medium. The shake flaskswere incubated at 37° C., 200 rpm for 7 days. Since the Microdochiumnivale carbohydrate oxidase contains FAD as a cofactor, one set offlasks also contained 52 μM riboflavin 5'-phosphate (Sigma Chemical Co.,St. Louis, Mo.).

Samples of 500 μl were removed at days 3, 5, and 7 from each flask andassayed for carbohydrate oxidase activity. Carbohydrate oxidase activitywas measured in a 96 well plate containing 10 μl of supernatant followedby the addition of 1 μl of o-anisidine, 69 μl of Britton and Robinsonbuffer pH 6.0, 10 μl of 1 M D-glucose, and 10 μl of Coprinus cinereusperoxidase (3.76 PODU/ml), obtained as described in WO 92/16634. Theactivity was measured at 405 nm for 10 minutes in mOD/min. Thetransformants all produced detectable carbohydrate oxidase activity. Theaddition of riboflavin 5'-phosphate to the shake flasks had a minoreffect on increasing activity. Samples of 20 μl from the highestcarbohydrate oxidase producers were run on an 8-16% Tris-Glycine gel(Novex, San Diego, Calif.) which confirmed the production ofcarbohydrate oxidase.

The transformants with the highest activities were spore purified bypatching isolated colonies onto new COVE plates twice in succession andthen regrown in shake flasks and retested for carbohydrate oxidaseactivity as above to confirm production of the carbohydrate oxidase.

Fermentations of Aspergillus oryzae JaL228 containing pEJG33 were run at34° C., pH 7, 1000-1200 rpm for 8 days in 2 liter lab fermentorscontaining medium composed of Nutriose, yeast extract, (NH₄)₂ HPO₄,MgSO₄.7H₂ O, citric acid, K₂ SO₄, CaCl₂.H₂ O, and trace metals solution.The trace metals solution (1000×) was composed per liter of 22 g ofZnSO₄.7H₂ O, 11 g of H₃ BO₃, 5 g of MnCl₂.4H₂ O, 5 g of FeSO₄.7H₂ O, 1.6g of CoCl₂.5H₂ O, 1.6 g of (NH₄)₆ Mo₇ O₂₄, and 50 g of Na₄ EDTA. Onefermentation was supplemented with 2×10⁻⁴ M FMN per liter (GOX003.8)while the other one was not (GOX002.8).

Eight day samples were assayed as described above. The results showedthe presence of carbohydrate oxidase activity in both fermentations, butno difference in carbohydrate oxidase activity was detected between thetwo fermentation broths.

Example 9

Expression of the Microdochium nivale Carbohydrate Oxidase Gene inFusarium venenatum

pEJG35 was introduced into Fusarium venenatum strain CC1-3 (WO 97/26330)using the method of Royer et al. (1995, Bio/Technology 13: 1479-1483)with BASTA™ (phosphinothricin resistance selection). The activeingredient in the herbicide BASTA™ is phosphinothricin. BASTA™ wasobtained from AgrEvo (Hoechst Schering, Rodovre, Denmark) and wasextracted twice with phenol:chloroform:isoamyl alcohol (25:24:1), andonce with chloroform:isoamyl alcohol (24:1) before use. Based on growthin the presence of BASTA™, 14 transformants were recovered and thengrown at room temperature on individual agar plates composed of 20 ml of5×Vogels medium (Royer et al., 1995, supra), 25 g of sucrose, 25 g ofnoble agar, and 25 mM NaNO₃ supplemented with mg of BASTA™ per ml. Thetransformants were then tested for carbohydrate oxidase production usingan indicator plate described by WiHeveen et al., 1990, supra. Fivetransformants tested positive. A plug from each positive transformantincluding untransformed Fusarium venenatum CC1-3 as a control wereinoculated into individual 125 ml shake flasks containing 30 ml ofM400Da medium supplemented with 0.5 g of CaCl₂ per liter and incubatedat 30° C., for 7 days under 150 rpm agitation. The M400Da medium wascomposed per liter of 50 g of maltodextrin, 2 g of MgSO₄, 2 g of KH₂PO₄, 4 g of citric acid, 2 g of urea, 0.5 g of CaCl₂, and 1 ml of Covetrace metals. One set of flasks also contained 52 μM riboflavin5'-phosphate.

At days 3, 5, and 7, 500 μl of culture broth was removed from each flaskand centrifuged. The supernatants were assayed for carbohydrate oxidaseactivity as described in Example 8. The transformants all produceddetectable carbohydrate oxidase activity. The addition of riboflavin5'-phosphate to the shake flasks had essentially no effect on increasingactivity. Samples of 20 μl from the highest carbohydrate oxidaseproducers were run on an 8-16% Tris-Glycine gel (Novex, San Diego,Calif.), which confirmed the production of carbohydrate oxidase. Thehighest producers were spore purified on Vogels/BASTA™ plates.

A fermentation of Fusarium venenatum strain CC1-3 containing pEJG35 wasrun at 30° C. for 8 days in a 2 liter fermentor containing mediumcomposed per liter of 20 g of sucrose, 2.0 g of MgSO₄.7H₂ O, 2.0 of KH₂PO₄, 2.0 of citric acid.H₂ O, 2.0 g of CaCl₂.2H₂ O, 0.5 ml of AMG tracemetals (pH adjusted to 4.5 prior to sterilization), and a filtersterilized mixture composed per liter of 2.5 g of urea and 30 ml of asoy vitamin mixture, which was added after sterilization and cooling ofthe medium. Feed streams were batched autoclaved mixtures composed ofsucrose and urea.

Eight day samples were assayed as described in Example 8. The resultsshowed the presence of carbohydrate oxidase activity.

Example 10

Purification of recombinant Microdochium nivale Carbohydrate Oxidase

The two Aspergillus oryzae JAL228 fermentation JaL228 broths, GOX002.8(1.2 l) and GOX003.8 (1.4 l), prepared as described in Example 8 werecombined. The combined broths were filtered using Whatman #2 filterpaper and then washed and concentrated by ultrafiltration to 512 mlusing an Amicon Spiral-Concentrator equipped with a S1Y10 membrane.Measurement of carbohydrate oxidase activity as described in Example 8indicated essentially 100% recovery of the enzyme.

The concentrate was then loaded onto a Q-Sepharose column (189 ml;Pharmacia Biotech, Inc., Piscataway, N.J.) pre-equilibrated with 10 mMTris-HCl pH 8. The carbohydrate oxidase was eluted with 2 M NaCl in 10mM Tris-HCl pH 8. Measurement of carbohydrate oxidase activity asdescribed above indicated most of the enzyme did not bind to the column.

The flow-through fraction (760 ml) from the Q-Sepharose column wasadjusted to pH 5.5 and loaded onto a SP-Sepharose column (176 ml;Pharmacia Biotech, Inc., Piscataway, N.J.) pre-equilibrated with 10 mMMES pH 5.5. The carbohydrate oxidase was eluted with 1 M NaCl in 10 mMMES pH 5.5, which yielded a carbohydrate oxidase preparation withapparent electrophoretic purity by SDS-PAGE. The table below summarizesthe purification of the recombinant carbohydrate oxidase. Overall a14-fold purification with a 31% recovery was achieved based on thefollowing oxygen electrode assay. Carbohydrate oxidase was measuredusing a Hansatech O₂ electrode with an assay solution composed of 0.26ml of 10 mM MES pH 5.5, 30 μl of 1.0 M D-glucose, and 3 μl ofcarbohydrate oxidase.

    __________________________________________________________________________    Purification of the recombinant carbohydrate oxidase                                 Vol A.sub.280                                                                        A.sub.280 × V                                                                A.sub.450                                                                        A.sub.450 × V                                                                A.sub.280 /A.sub.450                                                               Activity                                                                          Recovery                                  __________________________________________________________________________    Broth  2580                                                                              51.3                                                                             100  1.9                                                                              100  27   8.2 100                                         Ultrafiltration 512 44.2 17 2.35 25 19 45 107                                 Q- 768 20.7 12 1 16 21 24 87                                                  Sepharose                                                                     SP- 144 52.6 5.7 3.7 11 19 45 31                                              Sepharose                                                                   __________________________________________________________________________     Units: Vol, ml; A × V and Recovery (Activity × Vol), %;           Activity (Peroxidase/oanisidine assay), IU/ml.                           

Example 11

Molecular Properties of Recombinant Carbohydrate Oxidase

SDS-PAGE using a Novex 8-16% Tris-glycine SDS-PAGE gel indicated thatthe recombinant carbohydrate oxidases obtained as described in Examples8 and 9 have a molecular weight of approximately 55 kDa, similar to thewild-type carbohydrate oxidase.

N-terminal sequencing of the recombinant carbohydrate oxidases wasperformed on an Applied Biosystems 476A Protein Sequencer (PerkinElmer/Applied Biosystems Division, Foster City, Calif.) with on-lineHPLC and liquid phase trifluoroacetic acid (TFA) delivery. Therecombinant carbohydrate oxidases preparations were submitted toSDS-PAGE using Novex 10% Bis-Tris- SDS-PAGE gel using Novex Nupage MOPSbuffer under reducing conditions. The gels were transblotted to PVDFmembranes (Novex, San Diego, Calif.) for 2 hours at 25 volts in 10 mMCAPS pH 11.0 buffer. The PVDF membranes were stained in 0.1% CommassieBlue R250 in 40% methanol/1% acetic acid and the observed bands excised.The excised bands were sequenced from a blot cartridge using sequencingreagents (Perkin Elmer/Applied Biosystems Division, Foster City,Calif.). Detection of phenylthiohydantoin-amino acids was accomplishedby on-line HPLC using Buffer A containing 3.5% tetrahydrofuran in waterwith 18 ml of the Premix concentrate (Perkin Elmer/Applied BiosystemsDivision, Foster City, Calif.) containing acetic acid, sodium acetate,and sodium hexanesulfonate and Buffer B containing acetonitrile. Datawas collected and analyzed on a Macintosh IIsi using Applied Biosystems610 Data Analysis software.

N-terminal sequencing of the both excised bands produced the sequenceshown at positions 1-21 of SEQ ID NO: 2 where position 6 was notdeterminable but based on the deduced amino acid sequence is a cysteine.The N-terminus results agreed with the deduced amino acid sequence, andindicated a correct processing by both the Aspergillus oryzae andFusarium venenatum hosts.

In 10 mM MES-NaCl pH 5.5, the recombinant carbohydrate oxidase had aUV-visible spectrum typical for flavoproteins as recorded on a ShimadzuUV160U spectrophotometer with 1-cm quartz cuvette. The relativeabsorbance at 280 and 450 nm was 19, slightly larger than the 12 valueobtained for the wild-type enzyme. The extinction coefficient at 280 nmwas measured by amino acid analysis to be 1.9 g/(l×cm), whereas thepredicted value was 2.1 (including the contribution from a FADmolecule). Thus, it appeared that each recombinant carbohydrate oxidasecontained one flavin molecule (likely FAD).

Assuming that the oxidation of each D-glucose molecule was coupled tothe reduction of one O₂ to H₂ O₂, recombinant carbohydrate oxidaseactivity was measured using a Hansatech O₂ electrode as described inExample 10. The recombinant carbohydrate oxidase oxidized D-glucose (0.1M) at a specific activity of 4.0 IU/A₂₈₀ or 116 turnover/minute at pH5.5 and 20° C. As assayed by the Coprinus cinereus peroxidase/anisidinemethod described in Example 8, the recombinant carbohydrate oxidase hadthe same specific activity as wild-type enzyme.

Example 12

Substrate Specificity

Substrate Specificity at 60 mM Substrate Concentration

The substrate specificity for the carbohydrate oxidase from M. nivalewas determined in a microplate at ambient temperature by mixing in thefollowing order:

50 μl 0.4/0.4 M phosphate/citrate buffer (pH 6)

50 μl substrate (360 mM)

50 μl 21.6 mM 3-Dimethylaminobenzoic acid (DMAB)

50 μl 1 mM 3-Methyl-2-benzothiazolinone hydrazone (MBTH)

50 μl 75 μg/ml, rec. Coprinus cinereus peroxidase (rCiP)

50 μl carbohydrate oxidase

The absorbance at 595 nm was followed for at least 3 minutes. Theincrease in absorbance per minute was calculated and used as a measurefor relative activity.

The results of the oxidizing activity of the carbohydrate oxidase of thepresent invention on various mono- and disaccharide substrates aresummarized in the table below and show that the carbohydrate oxidase canoxidize most reducing sugars and shows higher activity on maltose andcellobiose than the corresponding monosaccharide glucose. The enzyme hadno activity on non-reducing sugars, such as fructose, sucrose,trehalose, and methyl-b-D-glucopyranoside. The results are shown assubstrate specificity of M. nivale oxidase, relative to the optimumactivity on D-cellobiose.

    ______________________________________                                        Substrate       & Activity                                                    ______________________________________                                        D-Glucose       69                                                              2-Deoxy-D-Glucose 4.2                                                         D-Galactose 31.3                                                              D-Mannose 3.2                                                                 D-Xylose 55.6                                                                 D-Maltose 83.5                                                                D-Cellobiose "100"                                                            Lactose 52.5                                                                ______________________________________                                    

Substrate Specificity at 0.83 mM

Further analyses of substrate specificity revealed that the carbohydrateoxidase from M. nivale is capable of oxidizing oligosaccharides of alldegrees of polymerization (DP) which were tested, DP2-DP5, using theassay conditions described above, except to change the substrateconcentration to 0.83 mM. Furthermore, the enzyme can hydrolyze bothmaltodextrins and cellodextrins wherein the monosaccharide units arelinked by alpha-1,4 or beta-1,4 glucosidic bonds, respectively. Thecarbohydrate oxidase hydrolyzed all cellodextrins tested equally welland at a level around 10-fold higher than the monosaccharide. Withmaltodextrins as the substrate, the activity of the carbohydrate oxidaseranged from 11/2-fold higher for maltohexaose to almost 5-fold higherfor maltotetraose than for the monosaccharide. The results aresummarized in the table below, showing the influence of the degree ofpolymerization and type of 1,4 linkage on carbohydrate oxidase activityrelative to DP 1 (D-glucose).

    ______________________________________                                                    % Activity                                                        DP            alpha-1.4                                                                              beta-1.4                                               ______________________________________                                        1             "100"                                                           2             211      949                                                      3 348 1147                                                                    4 477 1111                                                                    5 161 1014                                                                  ______________________________________                                    

Substrate Specificity at 1% Substrate Concentration

Analyses of the oxidizing activity of the carbohydrate oxidase from M.nivale on polysaccharides revealed that the enzyme is capable ofsignificant activity on carboxymethylcellulose (CMC), even after removalof smaller oligosaccharides and monosaccharides by dialysis when testedunder the assay conditions described above using a substrateconcentration of 1%. The results are summarized in the table below whichshows the carbohydrate oxidase activity on carboxymethylcelluloserelative to D-cellobiose.

    ______________________________________                                               Substrate                                                                              % Activity                                                    ______________________________________                                               D- cellobiose                                                                          "100"                                                           CMC 17.7                                                                      CMC, dialyzed  8.8                                                          ______________________________________                                    

Substrate Specificity at 10 mM Substrate Concentration

The oxidizing activity of the carbohydrate oxidase from M. nivale onvarious substrates was measured at pH 7.8 (50 mM Tris-HCl buffer), 10 mMof substrate. Similar data for carbohydrate oxidase from Acremonium areincluded for comparison (as described in BBA (1991) 1118, 41-47).

    ______________________________________                                                     Microdochium                                                                           Acremonium                                              ______________________________________                                        Maltose        76.1       100                                                   Maltotriose 84.9 94                                                           Maltotetraose 100.0 74                                                        Maltopentaose 58.6 46                                                         Maltohexaose 41.2 66                                                          Maltoheptaose 32.5 56                                                         Lactose 67.0 59                                                               Glucose 39.6 64                                                               Cellobiose 65.7 47                                                          ______________________________________                                    

Example 13

Oxidation of Maltose

To demonstrate that the reducing group at the 1-position is oxidized bythe carbohydrate oxidase from M. nivale, the oxidation of maltose wasfollowed chromatographically under the following conditions: 125 μl of0.2 M citrate-phosphate buffer, pH 6 was added to 250 μl 10 mM maltoseand 75 μl water before adding 50 μl purified M. nivale carbohydrateoxidase. The sample was incubated up to 30 minutes at 40° C. withconstant shaking. The reaction was stopped by adding 100 μl of thesample to 900 μl water at 95° C. 50 μl of the reaction mix was thenanalyzed by anion exchange chromatography (CarboPac PA1 column, Dionex)followed by pulsed amperometric detection (Dionex) on the Dionex DX-500system using the following conditions:

    ______________________________________                                        Flow rate:                                                                            1 ml/min                                                                A-buffer: 0.1 M NaOH (degassed in He)                                         B-buffer: 0.1 M NaOH, 0.6 mM sodium acetate (degassed in He)                  Gradient: 0-3 min, 5% B-buffer                                                 3-19 min, 5-30% B-buffer                                                      19-21 min, 30-100% B-buffer                                                   21-23 min, 100% B-buffer                                                      23-24 min 100-5% B-buffer                                                  ______________________________________                                    

Standard maltodextrins (DP1-7) were purchased from Sigma Chemical Co.Maltobionic acid was prepared according to Fitting & Putman (1952) J.Biol. Chem. 199:573.

Using the above method, maltose is detected as a peak with a retentiontime around 8.0 minutes, while maltobionic acid is detected as a peakwith a retention time around 13.3 minutes. At the above conditions,maltobionic acid is generated from maltose in the presence of the M.nivale carbohydrate oxidase, as a peak develops around 13.3 minutes.Thus, the carbohydrate oxidase oxidizes the free reducing end group inmaltose. The amount of maltobionic acid produced is presented below asμM maltobionic acid in reaction mixture:

    ______________________________________                                        Incubation time (minutes)                                                                      Maltobionic acid (μM)                                     ______________________________________                                        0                0                                                              5 150                                                                         10 370                                                                        30 880                                                                      ______________________________________                                    

Example 14

Binding Constant, K_(m)

Steady state kinetics was conducted by varying the concentration of thecarbohydrate substrates and determining the carbohydrate oxidaseactivity by the 4AA-TOPS assay. Simple Michaelis-Menten kinetics wasassumed although the reaction is not a simple one substrate-one productmechanism (E+S⃡ES→E+P).

The steady state kinetics for the carbohydrate oxidase from M. nivalewere investigated using some of the preferred substrates. Kineticconstants were obtained from a Lineweaver-Burke plot, assuming simpleMichaelis-Menten kinetics (although this is a rather poor assumption) toobtain apparent values "K_(m) " and "V_(max) " for various substrates.The results for "K_(m) " were:

    ______________________________________                                                Glucose:                                                                             42 mM                                                            Maltose: 11 mM                                                                Cellobiose 59 mM                                                            ______________________________________                                    

The carbohydrate oxidase shows the highest activity on cellobiose;however, "K_(m) " for cellobiose is almost 6 fold higher than formaltose. Likewise the "K_(m) " for glucose is significantly higher thanfor maltose, while the "V_(max) " is more or less the same for the twosubstrates. Thus, the previously shown preference for maltose at the lowconcentrations of substrate is explained by the lower value of "K_(m) "for maltose.

Example 15

pH and Temperature Activity Profiles

The activity of the carbohydrate oxidase from M. nivale over a pH rangewas determined in microplates at ambient temperature using the methoddescribed above in Example 12, but with buffers adjusted to the pH beingtested; the actual pH was measured in the reaction mixture. The resultsbelow (activity relative to the optimum at pH 6.32) show that thecarbohydrate oxidase has optimum activity at pH 5-7, and it has areasonably broad pH activity profile.

    ______________________________________                                               pH   % activity                                                        ______________________________________                                               3.38 5.58                                                                4.28 27.69                                                                    5.31 88.97                                                                    6.32 100.00                                                                   7.15 96.20                                                                    8.05 57.25                                                                  ______________________________________                                    

The temperature activity profile for the carbohydrate oxidase wasdetermined by mixing buffer and substrate in a glass tube andpreincubating at various temperatures (30-80° C.) for at least minutes:

150 ml 0.4/0.4 M phosphate/citrate pH 6

150 ml 180 mM maltose

150 ml oxidase dilution

Reactions were started by addition of oxidase and samples were incubatedat the appropriate temperature in a thermostatic bath, set at. After 5minutes the samples were placed on ice, and formation of H₂ O₂ wasdetermined by addition of 450 μl of DMAB:MBTH:rCiP (1:1:1) at therespective concentrations as in Example 12 and the increase inabsorbance at 590 nm was measured after 10 seconds on a HP 8452A diodearray spectrophotometer (Hewlett-Packard). A blind without incubationwas included. The results shown below (relative to the optimum at 50°C.) indicate that the enzyme is active up to at least 60° C. with anoptimum activity at 50° C.

    ______________________________________                                               ° C.                                                                        % activity                                                        ______________________________________                                               30   86.28                                                               40 90.98                                                                      50 100.00                                                                     60 79.95                                                                      70 2.27                                                                     ______________________________________                                    

Example 16

Thermostability by DSC

A sample of carbohydrate from M. nivale was desalted into 0.1 M MES, pH6 using the NAP-5 columns from Pharmacia. The sample (contianing 6.5mg/ml of the oxidase) was loaded onto the VP-DSC apparatus (MicroCal)and a linear scan from 20 to 90° C. was conducted at a scan rate of90°/h.

The denaturation temperature was found to be 73° C.

Temperature and pH Stability

Temperature Stability

The carbohydrate oxidase from M. nivale was pre-incubated 10 minutes atpH 6 at varying temperatures before measuring the residual activity bythe 4AA-TOPS

    ______________________________________                                        Temp ° C.:                                                                          Residual Activity %:                                             ______________________________________                                        40           81                                                                 50 78                                                                         60 100                                                                        70 19                                                                         80 2                                                                        ______________________________________                                    

The results shows that the enzyme is stable up to 60° C. but unstable at70° C. and above. This is in accordance with result obtained from DSCexperiments.

pH-stability

The carbohydrate oxidase from M. nivale was incubated for 2 hours at 40°C. at varying pH before measuring the residual activity by the 4AA-TOPSassay:

    ______________________________________                                                pH:  Res. Act. %:                                                     ______________________________________                                                3    2                                                                  4 100                                                                         5 95                                                                          6 93                                                                          7 99                                                                          8 97                                                                          9 93                                                                        ______________________________________                                    

The results shows that the enzyme is stable in the range from pH 4-9 butunstable at pH 3.

Example 18

Effect of Carbohydrate Oxidase on Gluten Rheology

    ______________________________________                                        Bread dough recipe                                                            ______________________________________                                        Wheat Flour    100% (= 10 g)                                                    Water  58% (including enzyme solution)                                        Salt  1.5%                                                                    Sugar  1.5%                                                                 ______________________________________                                    

The wheat flour was of the type Meneba. The flour was free of ascorbicacid.

The dough was mixed in a 10 g Micro Mixer (type NSI-33R, from NationalManufacturing Co.) for 2:30 minutes. Carbohydrate oxidase from M. nivalewas added before mixing. After mixing, the dough was allowed to rest for90 minutes at 32° C. and 85% relative humidity. Gluten was washed out ofthe dough with a solution of 2% NaCl (7 minutes in a Glutomatic 2200,Perten Instruments), and then centrifuged in a Gluten Index Centrifuge2015 (Perten Instruments) for 1 minute.

Gluten rheology was analyzed in a Bohlin VOR rheometer system (BohlinInstruments), performing a strain sweep at constant frequency (1 Hz), inorder to evaluate the strength of the dough under oscillation. In thismethod, the viscoelastic properties of the dough are divided into twocomponents, the dynamic shear storage modulus G' and the dynamic shearloss modulus G". The ratio of the loss and the storage moduli isnumerically equal to the tangent of the viscoelastic phase angle d. Anincrease in the storage modulus G' and a decrease in the phase angle dindicate a stronger and more elastic dough.

    ______________________________________                                        Carbohydrate Oxidase                                                                        G'          G"      d                                           ______________________________________                                        None (Reference)                                                                            349.5       166.4   25.46                                          50 U/kg flour 397.9 176.3 23.89*                                              100 U/kg flour 456.3* 193.7* 23.02*                                           200 U/kg flour 523.0* 207.4* 21.77*                                           500 U/kg flour 554.7* 207.3* 20.49*                                          1000 U/kg flour 708.5* 249.4* 19.40*                                        ______________________________________                                    

The results show that the G' storage modulus rises in proportion to thedose of carbohydrate oxidase added to the dough. With respect to the dphase angle, all the carbohydrate oxidase-treated doughs are differentfrom the reference, and the phase angle decreases proportionately withthe amount of enzyme added. Thus, the carbohydrate oxidase increases theelastic module, and hence increases dough elasticity, in adose-dependent manner. The figures are the average of three independentmeasurements. Figures denoted with an asterisk are statisticallysignificant from the reference by ANOVA analysis at a 5% level ofsignificance.

Example 19

Effect of Carbohydrate Oxidase on Dough Consistency

    ______________________________________                                        Baking Procedure                                                              ______________________________________                                        Basic recipe:                                                                          Flour (Meneba)                                                                            12 g (°100%)                                         Water 60% (including enzyme solution)                                         Yeast  4%                                                                     Sugar  1.5%                                                                   NaCl  1.5%                                                                 ______________________________________                                    

The flour was free of ascorbic acid.

Procedure: The dough was mixed in a 10 g Micro Mixer (type NSI-33R, fromNational Manufacturing Co.) for 21/2 minutes. Carbohydrate oxidase fromM. nivale was added before mixing. The final dough temperature aftermixing was approx. 27° C. The dough was evaluated immediately aftermixing.

Evaluation of Dough

Dough stickiness and firmness were measured empirically according to thefollowing scale:

    ______________________________________                                        Scoring                                                                         system 1 2 3 4 5 6                                                          ______________________________________                                        Firmness:                                                                            very soft                                                                              too soft soft/good                                                                            normal                                                                              firm too firm                             Stickiness: almost too sticky sticky good dry too dry                          liquid                                                                     ______________________________________                                    

The evaluation was conducted over two days, using three replicates foreach dose per day. The data, summarized in the table below, representthe mean value of six evaluations. The results indicate that both doughfirmness and stickiness show the same tendency of a dose-dependentincrease in the ability of the carbohydrate oxidase to yield a doughwith excellent dough consistency. At 200 and 300 Units/kg, a skilledbaker evaluated the dough as having an excellent firmness and softness,and a very airy consistency.

    ______________________________________                                        Carbohydrate Oxidase                                                                            Firmness Stickiness                                         ______________________________________                                        None (Reference)  3.0      2.3                                                   10 U/kg flour 3.0 3.0                                                         50 U/kg flour 3.5 3.4                                                        100 U/kg flour 4.0 4.0                                                        200 U/kg flour 4.0 3.8                                                        300 U/kg flour 4.1 4.1                                                        500 U/kg flour 4.8 4.8                                                        Bromate 4.0 3.5                                                             ______________________________________                                    

Example 20

In order to test the tolerance of Microdochium nivale carbohydrateoxidase (MCO) towards variations in different processing parameters, MCOhas been tested in a "European straight dough", standard scale bakingtest (2 kg flour). Compared to the standard baking procedure(ABF-SP.1217.01/01), the dough was strained by increasing the watercontent, by increasing the mixing time, and/or by increasing thefermentation time. The trial aimed to clarify if MCO can make the doughmore resistant to these changes. To simulate a realistic bakingprocedure MCO was tested in combination with Fungamyl Super MA (xylanaseand amylase). The set-up was based on a non-complete statistical design.Compared to a control, not containing MCO, the addition of MCO resultedin better dough/bread robustness. E.g. when evaluating the standing(=area of "foot") it can e.g. be concluded that no significantinteractions were observed between MCO dosage (0-200 U) and an increaseof the water addition by 1.5% and increase of the mixing time by +2 min.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 23                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 1553                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Microdochium nivale                                           <220> FEATURE:                                                                <221> NAME/KEY: intron                                                        <222> LOCATION: (1012)..(1076)                                                <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(1011)                                                   <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1077)..(1553)                                                <220> FEATURE:                                                                <221> NAME/KEY: mat.sub.-- peptide                                            <222> LOCATION: (67)..(1550)                                                   - - <400> SEQUENCE: 1                                                         - - atg cgt tct gca ttt atc ttg gcc ctc ggc ct - #t atc acc gcc agc        gct       48                                                                    Met Arg Ser Ala Phe Ile Leu Ala Leu Gly Le - #u Ile Thr Ala Ser Ala                  -20          - #       -15          - #       -10                      - - gac gct tta gtc act cgc ggt gcc atc gag gc - #c tgc ctg tct gct gct           96                                                                       Asp Ala Leu Val Thr Arg Gly Ala Ile Glu Al - #a Cys Leu Ser Ala Ala                -5             - # -1   1             - #  5                - #  10       - - ggc gtc ccg atc gat att cct ggc act gcc ga - #c tat gag cgc gat gtc          144                                                                       Gly Val Pro Ile Asp Ile Pro Gly Thr Ala As - #p Tyr Glu Arg Asp Val                            15 - #                 20 - #                 25              - - gag ccc ttc aac atc cgc ctg cca tac att cc - #c acc gcc att gct cag          192                                                                       Glu Pro Phe Asn Ile Arg Leu Pro Tyr Ile Pr - #o Thr Ala Ile Ala Gln                        30     - #             35     - #             40                  - - acg cag act act gct cac atc cag tcg gca gt - #c cag tgc gcc aag aag          240                                                                       Thr Gln Thr Thr Ala His Ile Gln Ser Ala Va - #l Gln Cys Ala Lys Lys                    45         - #         50         - #         55                      - - ctc aac ctc aag gtc tct gcc aag tct ggt gg - #t cac agc tac gcc tcg          288                                                                       Leu Asn Leu Lys Val Ser Ala Lys Ser Gly Gl - #y His Ser Tyr Ala Ser                60             - #     65             - #     70                          - - ttc ggc ttt ggt ggc gag aac ggt cac ctc at - #g gtc cag ctc gac cgc          336                                                                       Phe Gly Phe Gly Gly Glu Asn Gly His Leu Me - #t Val Gln Leu Asp Arg            75                 - # 80                 - # 85                 - # 90       - - atg att gat gtc atc tcg tac aat gac aag ac - #t ggc att gcc cat gtt          384                                                                       Met Ile Asp Val Ile Ser Tyr Asn Asp Lys Th - #r Gly Ile Ala His Val                            95 - #                100 - #                105              - - gag ccc ggt gcc cgc ctc gga cat ctc gcc ac - #c gtc ctc aac gac aag          432                                                                       Glu Pro Gly Ala Arg Leu Gly His Leu Ala Th - #r Val Leu Asn Asp Lys                       110      - #           115      - #           120                  - - tac ggc cgt gcc atc tcc cac ggt aca tgc cc - #t ggt gtc ggc atc tcc          480                                                                       Tyr Gly Arg Ala Ile Ser His Gly Thr Cys Pr - #o Gly Val Gly Ile Ser                   125          - #       130          - #       135                      - - ggc cac ttt gcc cac ggc ggc ttc ggc ttc ag - #c tcg cac atg cac ggt          528                                                                       Gly His Phe Ala His Gly Gly Phe Gly Phe Se - #r Ser His Met His Gly               140              - #   145              - #   150                          - - ctg gct gtc gac tcg gtc gtc ggt gtc act gt - #t gtt ctt gct gat gga          576                                                                       Leu Ala Val Asp Ser Val Val Gly Val Thr Va - #l Val Leu Ala Asp Gly           155                 1 - #60                 1 - #65                 1 -      #70                                                                              - - cgc atc gtt gag gct tct gcc act gag aat gc - #t gac ctc ttc tgg        ggt      624                                                                    Arg Ile Val Glu Ala Ser Ala Thr Glu Asn Al - #a Asp Leu Phe Trp Gly                          175  - #               180  - #               185              - - atc aag ggc gct ggc tcc aac ttc ggc atc gt - #t gct gtc tgg aag ctc          672                                                                       Ile Lys Gly Ala Gly Ser Asn Phe Gly Ile Va - #l Ala Val Trp Lys Leu                       190      - #           195      - #           200                  - - gcc act ttc cct gct ccc aag gtt ctc acc cg - #c ttt ggc gtc acc ctc          720                                                                       Ala Thr Phe Pro Ala Pro Lys Val Leu Thr Ar - #g Phe Gly Val Thr Leu                   205          - #       210          - #       215                      - - aac tgg aag aac aag acc tct gcc ctc aag gg - #c atc gag gct gtt gag          768                                                                       Asn Trp Lys Asn Lys Thr Ser Ala Leu Lys Gl - #y Ile Glu Ala Val Glu               220              - #   225              - #   230                          - - gac tac gcc cgc tgg gtc gcc ccc cgc gag gt - #c aac ttc cgc att gga          816                                                                       Asp Tyr Ala Arg Trp Val Ala Pro Arg Glu Va - #l Asn Phe Arg Ile Gly           235                 2 - #40                 2 - #45                 2 -      #50                                                                              - - gac tac ggc gct ggt aac ccg ggt atc gag gg - #t ctc tac tac ggc        act      864                                                                    Asp Tyr Gly Ala Gly Asn Pro Gly Ile Glu Gl - #y Leu Tyr Tyr Gly Thr                          255  - #               260  - #               265              - - ccc gag caa tgg cgt gcg gct ttc caa cct ct - #g ctc gac act ctg cct          912                                                                       Pro Glu Gln Trp Arg Ala Ala Phe Gln Pro Le - #u Leu Asp Thr Leu Pro                       270      - #           275      - #           280                  - - gct gga tac gtt gtc aac ccg acc acc tcc tt - #g aac tgg atc gag tcg          960                                                                       Ala Gly Tyr Val Val Asn Pro Thr Thr Ser Le - #u Asn Trp Ile Glu Ser                   285          - #       290          - #       295                      - - gtg ctc agc tac tcc aac ttt gac cat gtc ga - #c ttc att act cct cag         1008                                                                       Val Leu Ser Tyr Ser Asn Phe Asp His Val As - #p Phe Ile Thr Pro Gln               300              - #   305              - #   310                          - - cct gtaagtgttc accgactttg cgctgggaga atgttttatg tcggcttta - #c              1061                                                                       Pro                                                                           315                                                                            - - tgactccctc tacag gtc gag aac ttc tat gcc aag - #agc ttg acg ctc aag        1112                                                                                         Val - #Glu Asn Phe Tyr Ala Lys Ser Leu Thr Leu L - #ys                         - #               320  - #               325                 - - agt atc aag ggc gac gcc gtc aag aac ttt gt - #c gac tac tac ttt gac         1160                                                                       Ser Ile Lys Gly Asp Ala Val Lys Asn Phe Va - #l Asp Tyr Tyr Phe Asp                   330          - #       335          - #       340                      - - gtg tcc aac aag gtt aag gac cgc ttc tgg tt - #c tac cag ctc gac gtg         1208                                                                       Val Ser Asn Lys Val Lys Asp Arg Phe Trp Ph - #e Tyr Gln Leu Asp Val               345              - #   350              - #   355                          - - cac ggc ggc aag aac tcg caa gtc acc aag gt - #c acc aac gcc gag aca         1256                                                                       His Gly Gly Lys Asn Ser Gln Val Thr Lys Va - #l Thr Asn Ala Glu Thr           360                 3 - #65                 3 - #70                 3 -      #75                                                                              - - gcc tac cct cac cgc gac aag ctc tgg ctg at - #c cag ttc tac gac        cgc     1304                                                                    Ala Tyr Pro His Arg Asp Lys Leu Trp Leu Il - #e Gln Phe Tyr Asp Arg                          380  - #               385  - #               390              - - tac gac aac aac cag acc tac ccg gag acc tc - #a ttc aag ttc ctc gac         1352                                                                       Tyr Asp Asn Asn Gln Thr Tyr Pro Glu Thr Se - #r Phe Lys Phe Leu Asp                       395      - #           400      - #           405                  - - ggc tgg gtc aac tcg gtc acc aag gct ctc cc - #c aag tcc gac tgg ggc         1400                                                                       Gly Trp Val Asn Ser Val Thr Lys Ala Leu Pr - #o Lys Ser Asp Trp Gly                   410          - #       415          - #       420                      - - atg tac atc aac tac gcc gac ccc cgc atg ga - #c cgc gac tac gcc acc         1448                                                                       Met Tyr Ile Asn Tyr Ala Asp Pro Arg Met As - #p Arg Asp Tyr Ala Thr               425              - #   430              - #   435                          - - aag gtc tac tac ggt gag aac ctc gcc agg ct - #c cag aag ctc aag gcc         1496                                                                       Lys Val Tyr Tyr Gly Glu Asn Leu Ala Arg Le - #u Gln Lys Leu Lys Ala           440                 4 - #45                 4 - #50                 4 -      #55                                                                              - - aag ttt gat ccc acc gac cgt ttc tac tac cc - #t cag gct gtc cgc        cct     1544                                                                    Lys Phe Asp Pro Thr Asp Arg Phe Tyr Tyr Pr - #o Gln Ala Val Arg Pro                          460  - #               465  - #               470              - - gtc aaa taa              - #                  - #                       - #       1553                                                                  Val Lys                                                                        - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 495                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Microdochium nivale                                            - - <400> SEQUENCE: 2                                                         - - Met Arg Ser Ala Phe Ile Leu Ala Leu Gly Le - #u Ile Thr Ala Ser        Ala                                                                               1               5 - #                 10 - #                 15             - - Asp Ala Leu Val Thr Arg Gly Ala Ile Glu Al - #a Cys Leu Ser Ala Ala                   20     - #             25     - #             30                  - - Gly Val Pro Ile Asp Ile Pro Gly Thr Ala As - #p Tyr Glu Arg Asp Val               35         - #         40         - #         45                      - - Glu Pro Phe Asn Ile Arg Leu Pro Tyr Ile Pr - #o Thr Ala Ile Ala Gln           50             - #     55             - #     60                          - - Thr Gln Thr Thr Ala His Ile Gln Ser Ala Va - #l Gln Cys Ala Lys Lys       65                 - # 70                 - # 75                 - # 80       - - Leu Asn Leu Lys Val Ser Ala Lys Ser Gly Gl - #y His Ser Tyr Ala Ser                       85 - #                 90 - #                 95              - - Phe Gly Phe Gly Gly Glu Asn Gly His Leu Me - #t Val Gln Leu Asp Arg                  100      - #           105      - #           110                  - - Met Ile Asp Val Ile Ser Tyr Asn Asp Lys Th - #r Gly Ile Ala His Val              115          - #       120          - #       125                      - - Glu Pro Gly Ala Arg Leu Gly His Leu Ala Th - #r Val Leu Asn Asp Lys          130              - #   135              - #   140                          - - Tyr Gly Arg Ala Ile Ser His Gly Thr Cys Pr - #o Gly Val Gly Ile Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly His Phe Ala His Gly Gly Phe Gly Phe Se - #r Ser His Met His        Gly                                                                                             165  - #               170  - #               175             - - Leu Ala Val Asp Ser Val Val Gly Val Thr Va - #l Val Leu Ala Asp Gly                  180      - #           185      - #           190                  - - Arg Ile Val Glu Ala Ser Ala Thr Glu Asn Al - #a Asp Leu Phe Trp Gly              195          - #       200          - #       205                      - - Ile Lys Gly Ala Gly Ser Asn Phe Gly Ile Va - #l Ala Val Trp Lys Leu          210              - #   215              - #   220                          - - Ala Thr Phe Pro Ala Pro Lys Val Leu Thr Ar - #g Phe Gly Val Thr Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn Trp Lys Asn Lys Thr Ser Ala Leu Lys Gl - #y Ile Glu Ala Val        Glu                                                                                             245  - #               250  - #               255             - - Asp Tyr Ala Arg Trp Val Ala Pro Arg Glu Va - #l Asn Phe Arg Ile Gly                  260      - #           265      - #           270                  - - Asp Tyr Gly Ala Gly Asn Pro Gly Ile Glu Gl - #y Leu Tyr Tyr Gly Thr              275          - #       280          - #       285                      - - Pro Glu Gln Trp Arg Ala Ala Phe Gln Pro Le - #u Leu Asp Thr Leu Pro          290              - #   295              - #   300                          - - Ala Gly Tyr Val Val Asn Pro Thr Thr Ser Le - #u Asn Trp Ile Glu Ser      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Val Leu Ser Tyr Ser Asn Phe Asp His Val As - #p Phe Ile Thr Pro        Gln                                                                                             325  - #               330  - #               335             - - Pro Val Glu Asn Phe Tyr Ala Lys Ser Leu Th - #r Leu Lys Ser Ile Lys                  340      - #           345      - #           350                  - - Gly Asp Ala Val Lys Asn Phe Val Asp Tyr Ty - #r Phe Asp Val Ser Asn              355          - #       360          - #       365                      - - Lys Val Lys Asp Arg Phe Trp Phe Tyr Gln Le - #u Asp Val His Gly Gly          370              - #   375              - #   380                          - - Lys Asn Ser Gln Val Thr Lys Val Thr Asn Al - #a Glu Thr Ala Tyr Pro      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - His Arg Asp Lys Leu Trp Leu Ile Gln Phe Ty - #r Asp Arg Tyr Asp        Asn                                                                                             405  - #               410  - #               415             - - Asn Gln Thr Tyr Pro Glu Thr Ser Phe Lys Ph - #e Leu Asp Gly Trp Val                  420      - #           425      - #           430                  - - Asn Ser Val Thr Lys Ala Leu Pro Lys Ser As - #p Trp Gly Met Tyr Ile              435          - #       440          - #       445                      - - Asn Tyr Ala Asp Pro Arg Met Asp Arg Asp Ty - #r Ala Thr Lys Val Tyr          450              - #   455              - #   460                          - - Tyr Gly Glu Asn Leu Ala Arg Leu Gln Lys Le - #u Lys Ala Lys Phe Asp      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Pro Thr Asp Arg Phe Tyr Tyr Pro Gln Ala Va - #l Arg Pro Val Lys                         485  - #               490  - #               495              - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                               <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                               <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (3)                                                           <223> OTHER INFORMATION: i                                                    <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (6)                                                           <223> OTHER INFORMATION: i                                                    <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (9)                                                           <223> OTHER INFORMATION: i                                                    <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (12)                                                          <223> OTHER INFORMATION: i                                                    <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (15)                                                          <223> OTHER INFORMATION: i                                                     - - <400> SEQUENCE: 3                                                         - - gcngcnggng tnccnathga yat           - #                  - #                    23                                                                      - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                               <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: i                                                    <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (7)                                                           <223> OTHER INFORMATION: i                                                     - - <400> SEQUENCE: 4                                                         - - nggrtcngcr tarttdatrt acat          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 5                                                         - - tccagttcta cgaccgctac g           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 6                                                         - - cagacttggc agagaccttg a           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                               <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (1)                                                           OTHER INFORMATION:     i                                                      <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (19)                                                          <223> OTHER INFORMATION: i                                                     - - <400> SEQUENCE: 7                                                         - - nacrtcraar tartartcna craartt          - #                  - #                 27                                                                      - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 15                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                               <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (4)                                                           <223> OTHER INFORMATION: i                                                    <220> FEATURE:                                                                <221> NAME/KEY: modified.sub.-- base                                          <222> LOCATION: (10)                                                          <223> OTHER INFORMATION: i                                                     - - <400> SEQUENCE: 8                                                         - - rttnacccan ccrtc              - #                  - #                      - #    15                                                                   - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 9                                                         - - nggrtcngcr tarttdatrt acat          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 10                                                        - - datraartcn acrtgrtcra artt          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 11                                                        - - ccaytgytcn ggngtnccrt arta          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 12                                                        - - ctcgccactt tccctgctcc c           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 13                                                        - - ctcggtcacc aaggctctcc c           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 14                                                        - - gaccgctacg acaacaacca g           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 15                                                        - - tcggagaaat gagagcaacc a           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 16                                                        - - agccgacgtc cagcatagca g           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 17                                                        - - accctaccat acgagttcac g           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 18                                                        - - ggtcgaatcg tcacaaagta t           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 19                                                        - - cactggactg ccgactggat g           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 20                                                        - - caacaaccag acctaccc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 21                                                        - - ctcagcagca cttcttttca t           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 30                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 22                                                        - - gatttaaata tgcgttctgc atttatcttg         - #                  - #               30                                                                      - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 30                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 23                                                        - - gttaattaat tatttgacag ggcggacagc         - #                  - #               30                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated carbohydrate oxidase selected fromthe group consisting of:(a) a polypeptide encoded by the carbohydrateoxidase-encoding part of the DNA sequence cloned into a plasmid presentin Escherichia coli NRRL B-30034, wherein the plasmid is pCR2.1-TOPO;(b) a polypeptide having an amino acid sequence as shown in SEQ ID NO:2, or a fragment thereof exhibiting carbohydrate oxidase activity; and(c) a polypeptide having at least 90% identity to the polypeptide of(a)-(b) and exhibiting carbohydrate oxidase activity.
 2. The isolatedcarbohydrate oxidase of claim 1, wherein the polypeptide is produced byMicrodochium nivale CBS
 100236. 3. The isolated carbohydrate oxidase ofclaim 1 selected from (a) or (b).
 4. A dough-improving additivecomprising:a) the carbohydrate oxidase of claim 1 which has has a higheractivity on an oligosaccharide substrate having a degree ofpolymerization of 2 or higher relative to its activity on thecorresponding monosaccharide; and b) a second enzyme selected from thegroup consisting of amylase, cellulose, hemicellulase, lipase andphospholipase.
 5. The composition of claim 4 wherein the second enzymeis an amylase which hydrolyzes starch to form oligosaccharides as a mainproduct.
 6. The composition of claim 5 wherein the amylase is selectedfrom the group consisting of a Bacillus stearothermophilus maltogenicalpha-amylase, an Aspergillus oryzae alpha-amylase, and a beta-amylase.7. A dough-improving additive, which comprises the carbohydrate oxidaseof claim 1 which has a higher activity on an oligosaccharide substratehaving a degree of polymerization of 2 or higher relative to itsactivity on the corresponding monosaccharide, wherein said additive isin the form of a granulate or agglomerated powder.
 8. The additive ofclaim 7 wherein more than 95% of said additive has a particle sizebetween 25 and 500 μm.
 9. The carbohydrate oxidase of claim 1, whereinsaid carbohydrate oxidase is produced by Microdochium nivale.
 10. Amethod for producing the carbohydrate oxidase of claim 1, said methodcomprising (i) cultivating a carbohydrate oxidase producing strain ofMicrodochium in a suitable nutrient medium, and (ii) recovering thecarbohydrate oxidase.
 11. The method claim 10, wherein said strain is astrain of M. nivale.
 12. A carbohydrate oxidase which is derived from astrain of Microdochium and has an oxidizing activity on maltotetraose asa substrate which is at least twice as much as its oxidizing activity onglucose as a substrate, when said activities are measured at a substrateconcentration of 0.83 mM.
 13. The carbohydrate oxidase of claim 12 whichis derived from a strain of M. nivale.